INFLUENCE OF BLOOD METABOLITE CONCENTRATIONS ON THE RESUMPTION OF POSTPARTUM OVARIAN FUNCTION IN FRIESIAN-SANGA CROSSBRED COWS BY COURAGE MAcCARTHY VUVOR (10444071) THIS THESIS IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL ANIMAL SCIENCE DEGREE DEPARTMENT OF ANIMAL SCIENCE COLLEGE OF BASIC AND APPLIED SCIENCES UNIVERSITY OF GHANA JULY, 2015 University of Ghana http://ugspace.ug.edu.gh i DECLARATION I, COURAGE MAcCARTHY VUVOR, author of this thesis entitled “Influence of blood metabolite concentrations on the resumption of postpartum ovarian function in Friesian- Sanga cows” do declare that except for references to other people’s work, the work presented in this thesis was done entirely by me in the Department of Animal Science, University of Ghana, Legon from August, 2013 to July, 2015. This work has never been submitted in whole or in part for any degree in this University or elsewhere. …………………………………. COURAGE MAcCARTHY VUVOR This work has been submitted for examination with our approval as supervisors: ………………………………….. DR. FREDERICK YEBOAH OBESE (MAJOR SUPERVISOR) …………………………………. DR. RICHARD OSEI-AMPONSAH (CO-SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION I dedicate this work to my mother, Mrs. Charity Beatrice Afadzinu-Vuvor, my siblings, Rapheal Gyane, Steven Amoako, Cosmos Kwashigah Vuvor, Mrs. Precious Emefah Konu, and Charity Lois Eyram Vuvor, and to the SOSA family. University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS I give gratitude to the Almighty God for His guidance, grace, inspiration, and strength throughout my study period. I am very much obligated to my supervisors Dr. Frederick Yeboah Obese and Dr. Richard Osei-Amponsah of the Department of Animal Science, University of Ghana, Legon for their critique and priceless suggestions towards the success of this research. I want to appreciate the huge contribution of my mother, Mrs. Charity Beatrice Afadzinu- Vuvor, my siblings, Rapheal Gyane, Steven Amoako, Cosmos Kwashigah Vuvor, Mrs. Precious Emefah Konu, and Charity Lois Eyram Vuvor. I also want to express my sincere gratitude to the SOSA family for their support and encouragement during the course of this work. Special thanks also go to the Department of Animal Science of the University of Ghana, the Head of Department, senior members, and office staff for their huge contributions, and motivation to ensure that this work is completed. I acknowledge the assistance of Mr. Mama Abdulai, Mr. Afranie and Ms. Doreen Owusu Ntumy of the Animal Research Institute – Council for Scientific and Industrial Research in the collection of blood samples for the study. Special thanks go to Mr. Tagoe of the Biochemistry Department and Mr. Ofori of the Central Laboratory of the Korle-Bu teaching Hospital for their assistance in determination of progesterone and blood metabolites. I acknowledge the assistance provided by the Physiology Laboratory of Graeme Martin, School of Animal Biology, Faculty of Agriculture and Natural Resources, the University of Western Australia, for measurement of the metabolic hormones, insulin, insulin-like growth factor-I and leptin. University of Ghana http://ugspace.ug.edu.gh iv Finally, I am sincerely grateful to Mr. Rapheal Ayizanga of the Department of Animal Science, University of Ghana for his assistance in statistical analysis of the data obtained in this study. University of Ghana http://ugspace.ug.edu.gh v TABLE OF CONTENTS DECLARATION ........................................................................................................................ i DEDICATION ........................................................................................................................... ii ACKNOWLEDGEMENTS ..................................................................................................... iii TABLE OF CONTENTS ........................................................................................................... v LIST OF TABLES .................................................................................................................. ixx LIST OF FIGURES ................................................................................................................... x LIST OF PLATES.................................................................................................................... xi ABSTRACT ............................................................................................................................. xii LIST OF ACRONYMS .......................................................................................................... xiv CHAPTER ONE ........................................................................................................................ 1 INTRODUCTION ..................................................................................................................... 1 1.1 BACKGROUND AND JUSTIFICATION ...................................................................... 1 1.2 OBJECTIVES .................................................................................................................. 3 CHAPTER TWO ....................................................................................................................... 4 LITERATURE REVIEW .......................................................................................................... 4 2.1 OESTRUS CYCLE IN CATTLE .................................................................................... 4 2.2 POSTPARTUM ANOESTROUS IN CATTLE .............................................................. 5 2.3 FACTORS AFFECTING POSTPARTUM ANOESTROUS INTERVAL ..................... 6 2.3.1 Effect of nutrition on postpartum anoestrous interval ............................................... 6 University of Ghana http://ugspace.ug.edu.gh vi 2.3.2 Effect of suckling on postpartum anoestrous interval ............................................... 8 2.3.3 Effect of season on postpartum anoestrous interval ................................................ 10 2.3.4 Effect of breed and genotype on postpartum anoestrous interval ........................... 11 2.3.5 Effect of Age and parity on postpartum anoestrous interval ................................... 11 2.3.6 Effect of peri-paturient diseases on postpartum anoestrous interval ....................... 12 2.3.7 Effect of milk yield on postpartum anoestrous interval .......................................... 13 2.4 METABOLIC HORMONES AND NUTRITIONAL METABOLITES ASSOCIATED WITH OVARIAN FUNCTION IN CATTLE ..................................................................... 14 2.4.1 Role of growth hormone (GH) in ovarian function in cattle ................................... 14 2.4.2 Role of insulin-like growth factor-I (IGF-I) in ovarian function in cattle ............... 15 2.4.3 Role of insulin in ovarian function in cattle ............................................................ 16 2.4.4 Role of leptin in ovarian function in cattle .............................................................. 17 2.4.5 Role of glucose in ovarian function in cattle ........................................................... 18 2.4.6 The role of non-esterified fatty acids (NEFAs) in ovarian function in cattle .......... 19 2.4.7 Role of cholesterol in ovarian function in cattle ...................................................... 20 2.4.8 Role of proteins in ovarian function in cattle .......................................................... 20 2.4.9 Role of blood urea nitrogen in ovarian function in cattle ........................................ 22 2.4.10 Role of creatinine in ovarian function in cattle ..................................................... 22 CHAPTER TREE .................................................................................................................... 24 MATERIALS AND METHODS ............................................................................................. 24 University of Ghana http://ugspace.ug.edu.gh vii 3.1 Location of study ............................................................................................................ 24 3.2 Management of Animals ................................................................................................ 24 3.3 Blood sampling for progesterone, metabolic hormones and nutritional metabolites ..... 26 3.4 Measurement of progesterone, metabolic hormones and nutritional metabolite concentrations....................................................................................................................... 26 3.4.1 Progesterone Assay.................................................................................................. 27 3.4.2 Growth hormone (GH) Assay .................................................................................. 29 3.4.3 IGF-I Assay ............................................................................................................ 30 3.4.4 Insulin Assay .......................................................................................................... 31 3.4.5 Nutritional metabolite analyses ............................................................................... 32 3.5 Statistical Analysis ......................................................................................................... 33 CHAPTER FOUR .................................................................................................................... 35 RESULTS ................................................................................................................................ 35 4.1 Resumption of ovarian activity ..................................................................................... 35 4.2 Daily milk yield, BW and BCS in early-cycling, late-cycling and non-cycling cows . 35 4.3 Metabolic hormone concentrations in early-cycling, late-cycling and non-cycling cows .............................................................................................................................................. 37 4.4 Nutritional metabolite concentrations in early-cycling, late-cycling and non-cycling cows ...................................................................................................................................... 42 4.5 Correlations among metabolic hormones and nutritional metabolites ........................... 49 University of Ghana http://ugspace.ug.edu.gh viii CHAPTER FIVE ..................................................................................................................... 51 DISCUSSION .......................................................................................................................... 51 5.1 Resumption of ovarian activity in Friesian-Sanga crossbred cows................................ 51 5.2 Daily milk yield, BW and BCS and resumption of ovarian activity ............................ 52 5.3 Metabolic hormones concentrations and resumption of ovarian activity ...................... 53 5.4 Nutritional metabolite concentrations and resumption of ovarian activity .................... 54 5.5 Relationships among metabolic hormones and nutritional-related metabolites............. 56 CHAPTER SIX ........................................................................................................................ 58 CONCLUSIONS AND RECOMMENDATIONS .................................................................. 58 6.1 CONCLUSIONS ............................................................................................................ 58 6.2 RECOMMENDATIONS .............................................................................................. 59 REFERENCES ........................................................................................................................ 60 APPENDICES ......................................................................................................................... 82 University of Ghana http://ugspace.ug.edu.gh ix LIST OF TABLES Table 4.1. Resumption of ovarian activity in Friesian-Sanga crossbred cows ......................35 Table 4.2. Milk yield, body weight and body condition score in ovarian activity groups in Friesian-Sanga crossbred cattle................................................................................................ 36 Table 4.3. Postpartum plasma hormone metabolite concentrations in early-cycling, late cycling and non-cycling Friesian-Sanga crossbred cows ........................................................ 38 Table 4.4. Postpartum plasma metabolite concentrations in early-cycling, late cycling and non- cycling Friesian-Sanga crossbred cows ........................................................................... 42 Table 4.5. Partial Correlation coefficients among plasma concentrations of GH, IGF-I, insulin, glucose, cholesterol, total protein, albumin, globulin, urea and creatinine in Friesian- Sanga crossbred cows(n=16) during weeks 1, 3, 5, 7, and 9 postpartum ................................ 50 University of Ghana http://ugspace.ug.edu.gh x LIST OF FIGURES Figure 4.1: Changes in milk yield in early-cycling, late-cycling and non-cycling Friesian- Sanga crossbred cows during the first 10 weeks postpartum .................................................. 37 Figure 4.2: Changes in plasma IGF-I concentrations in early-cycling, late-cycling and non- cycling Friesian-Sanga crossbred cows during the first 10 weeks postparartum. ................... 39 Figure 4.3: Changes in plasma insulin concentrations in early-cycling, late-cycling and non- cycling Friesian-Sanga crossbred cows during the first 10 weeks postpartum. ..................... 40 Figure 4.4: Changes in plasma insulin concentrations in early-cycling, late-cycling and non- cycling Friesian-Sanga crossbred cows during the first 10 weeks postpartum ....................... 41 Figure 4.5: Changes in plasma concentration of glucose in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum .................. 43 Figure 4.6: Changes in plasma concentration of Total protein in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum ........... 44 Figure 4.7: Changes in plasma concentration of globulin in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum. ................. 45 Figure 4.8: Changes in plasma concentration of urea in early-cycling, late-cycling and non- cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum. ........................ 46 Figure 4.9: Changes in plasma concentration of creatinine in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum. ................. 47 Figure 4.10: Changes in plasma concentration of cholesterol in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum. .......... 48 University of Ghana http://ugspace.ug.edu.gh xi Figure 4.11: Changes in plasma concentration of albumin in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows during the first 9 weeks postpartum. ................. 49 LIST OF PLATES Plate 3.1: Friesian-Sanga cross-bred cow……………………………………………….... .25 Plate 3.2: Packard Cobra-II Auto Gamma Counter ................................................................ 30 Plate 3.3: URIT 810 Semi auto Biochemical Analyzer......................................................... 33 University of Ghana http://ugspace.ug.edu.gh xii ABSTRACT An experiment was conducted to investigate the effect of plasma concentrations of the metabolic hormones [Growth hormone (GH), insulin and insulin-like growth factor –I (IGFI)] and nutritional metabolites (glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) on the resumption of postpartum ovarian activity in sixteen Friesian-Sanga crossbred cows at the Animal Research Institute’s Frafraha station. Also, correlation among the metabolic hormones and the nutritional metabolites were assessed. The cows grazed extensively on natural grassland without any feed supplementation. They were weighed monthly and scored for body condition once every week using the 9-point score (1= very thin to 9 = obese). Blood samples were taken from cows from week 1 to 16 postpartum and processed for plasma. The concentrations of the metabolic hormones (GH, insulin and IGF-I in the plasma) were measured weekly from week 1 to 10 postpartum, whilst the nutritional metabolites (glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) were determined at two-weekly intervals (weeks 1, 3, 5, 7 and 9). Resumption of postpartum ovarian cyclicity was determined by measuring progesterone concentration in the plasma from week 1 to 16 in the cows. Cows were classified as having resumed ovarian activity when a plasma progesterone concentration of ≥ 1.0 ng/ml was recorded for two consecutive weekly samples. Based on the resumption of ovarian activity, cows were classified as early- cycling, late-cycling or non-cycling. Results from the present study indicate that 37.5% of cows commenced ovarian cyclicity early (by 56 days postpartum), 37.5 % commenced ovarian activity late (within 57-112 days postpartum), while 25 % failed to commence ovarian cyclicity by 112 days (16 weeks) postpartum. Partial milk yield, body condition score or body weight was not significantly different (P>0.05) in the three ovarian activity groups. The concentrations of the metabolic hormones, GH and insulin were similar (P>0.05) in the three ovarian activity groups, likewise the concentrations of the nutritional metabolites, University of Ghana http://ugspace.ug.edu.gh xiii glucose, total protein, globulin, urea and creatinine. Plasma IGF-I concentration was higher (P<0.001) in early cycling (18.7 +0.74 ng/mL) than in late-cycling (12.4 + 0.75 ng/mL) and non-cycling (10.4 + 0.91 ng/mL) cows. Plasma cholesterol concentration was significantly lower (P < 0.05) in early-cycling (1.94 + 0.15 mmol/L) compared with late-cycling (2.48 + 0.12 mmol/L) and non-cycling (2.61 + 0.11 mmol/L) cows. For plasma albumin concentrations, the levels recorded for early-cycling cows were higher (40.7 + 2.85 g/L) than in late-cycling (34.4 + 1.97 g/L) and non-cycling (33.6 + 2.66 g/L) cows. There was a positive correlation between IGF-I and insulin (r = 0.328; P<0.01); glucose (r = 0.260; P<0.05), and cholesterol (r = 0.262; P<0.05), while insulin was positively correlated with glucose (r = 0.502; P<0.10). Glucose was negatively correlated with cholesterol (r = - 0.264; P<0.05), and globulin (r= -0.323; P<0.01); but positively correlated with albumin (r = 0.291; P<0.05). Total protein was positively correlated with globulin (r = 0.706; P<0.01), and urea (r = 0.442; P<0.01). Albumin was negatively correlated with globulin (r = -0.654; P<0.01), and globulin was positively correlated with urea (r = 0.267; P<0.05). The results from the study suggest poor nutritional and metabolic status of cows. Higher plasma concentrations of IGF-I in the early postpartum period was associated with early resumption of ovarian cyclicity in cows. Also, cows with lower plasma concentrations of albumin, but higher plasma concentrations of cholesterol were at risk of delayed resumption of postpartum ovarian activity. Introducing feed supplementation strategies during the postpartum period should positively influence the synthesis and secretion of metabolic hormones associated with resumption of ovarian cyclicity and improve the reproductive performance of the Friesian-Sanga crossbred cows. University of Ghana http://ugspace.ug.edu.gh xiv LIST OF ACRONYMS BCS Body condition score BHB Beta-hydroxybutyrate BW Body weight FSH follicle-stimulating hormone GH Growth hormone GnRH Gonadotrophin-releasing hormone IGF-I Insulin-like growth factor-I LH Luteinizing hormone NEFA Non-esterified fatty acids University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE INTRODUCTION 1.1 BACKGROUND AND JUSTIFICATION One of the major factors that influences the operations and profitability of cattle production is reproductive performance of the animals (Wetteman et al., 2003). Ovarian function in cattle is regulated by important factors, including nutrition and body condition (Montiel and Ahuja, 2005; Roche et al., 2007); thus the loss of body condition at calving and during the postpartum period has been associated with delays in resumption of ovarian cycles (Shrestha et al., 2005; Soca et al., 2014). Also, high nutrient requirements for milk production during early lactation induces a physiological state of negative energy balance in most cows (Walsh et al., 2011; Esposito et al., 2014), and this tends to delay ovulation in cows (Butler, 2000; Butler, 2005). Energy balance is the difference between the dietary intake of utilizable energy and the expenditure of energy for body maintenance, growth and milk production (Beam and Butler, 1999). Blood metabolites include metabolic hormones such as growth hormone (GH), insulin, insulin-like growth factor-I (IGF-I), thyroid hormones (thyroxine and triiodothyronine), leptin, and nutritional metabolites such as non-esterified fatty acids (NEFA), beta- hydroxybutyrate (BHB), glucose, cholesterol, total protein, albumin, globulin and urea. These metabolites are involved in energy and protein homeostasis, and reproductive function, especially the resumption of ovulation in cows during the postpartum period (Diskin et al., 2003; Konigsson et al., 2008). For example, increased concentrations of IGF-I, insulin and glucose in the postpartum period have been associated with early resumption of ovarian University of Ghana http://ugspace.ug.edu.gh 2 cycles in cows (Beam and Butler, 1997; Samadi et al., 2013), while elevated concentrations of NEFA and BHB have been associated with delays in the resumption of ovarian activity (Reist et al., 2000; Hess et al., 2005; Giuliodori et al., 2011). In Ghana, crossbreeding programmes involving exotic and indigenous breeds have been embarked upon in a bid to improve meat and milk yield, and reproductive performance. A common example is the cross between the exotic Holstein-Friesian and the local Sanga to evolve a dual-purpose breed (Friesian-Sanga) for meat and milk production (Obese et al., 2013). The Friesian-Sanga crossbred cattle may however be exposed to nutritional deficiencies as a result of extensive grazing on natural pasture which often become scarce and poor in quality, especially during the dry season. The dry matter intake of cows managed in a pasture-based system is often less than in confined systems in which cows are fed total mixed rations. Thus pasture based diets may provide insufficient nutrients to sustain high milk and meat production. This situation is likely to be aggravated during the early postpartum period when nutritional demands are high, thus adversely affecting the concentrations of metabolic hormones and nutritional metabolites in the blood associated with reproductive function in cows. The early resumption of ovarian cycles following calving is necessary for the maintenance of yearly calving intervals necessary for high reproductive efficiency in cattle production (Mihm, 1999). Obese et al. (2009) indicated that delays in the resumption of ovarian activity postpartum in indigenous cows in Ghana (Sanga, West African Shorthorn, and N’dama) have contributed to extended calving intervals causing financial loss to herd owners. The authors identified nutritional deficiencies and prolonged suckling of cows by calves as the major factors delaying the resumption of ovarian activity through inhibition of secretion of University of Ghana http://ugspace.ug.edu.gh 3 metabolic hormones and nutritional metabolites involved with ovarian follicular development and function. From the above, metabolic hormones and nutritional metabolites could mediate the effect of nutrient intake on reproduction, as suggested by Wetteman and Bossis (2000). Information is however, limited regarding the relationships among concentrations of metabolic hormones, nutritional metabolites in the blood, and resumption of postpartum ovarian activity in Friesian-Sanga crossbred cows managed within the pasture-based systems in Ghana. Such information is critical in providing an understanding of the impact of nutritional and metabolic status on reproduction which could guide the development of management strategies that foster an early resumption of ovarian activity postpartum. 1.2 OBJECTIVES The objectives of this study were to: i) Determine the plasma concentrations of some metabolic hormones (GH, IGF-I, insulin), and nutritional metabolites (glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) which are associated with energy and protein homeostasis in Friesian- Sanga cows during early lactation. ii) Assess the relationships between plasma concentrations of metabolites (metabolic hormones and nutritional metabolites) and the resumption of ovarian activity in Friesian- Sanga cows during early lactation, and iii) Determine the correlations among the plasma concentrations of the metabolites in Friesian-Sanga cows during early lactation. University of Ghana http://ugspace.ug.edu.gh 4 CHAPTER TWO LITERATURE REVIEW 2.1 OESTRUS CYCLE IN CATTLE Females of domestic animals come into heat (oestrus) at fairly regular intervals that differ widely between species (Hafez and Hafez, 2000). The oestrus cycle is defined as the time interval between periods of oestrus (Bearden and Fuquay, 1984; Frandson et al., 2009). The length of the oestrus cycle in the cow ranges from 21-22 days (Hafez and Hafez, 2000), and consists of two main events (i) those associated with the growth of ovarian follicles (follicular phase lasting 3 days), comprising pro-oestrus and oestrus, and (ii) those associated with the growth of the corpus luteum (luteal phase lasting 18 days) which comprises met- oestrus and di-oestrus (Morrow, 1986; Frandson et al., 2009). The pro-oestrus period is characterized by growth of ovarian follicles and oestrogen production under the influence of follicle stimulating hormone (FSH) from the anterior pituitary gland (Frandson et al., 2009). Oestrus is the period when the female is receptive to the male and will stand for mating (Bearden and Fuquay, 1984; Frandson et al., 2009). Behavioural signs of oestrus are due to the influence of oestrogens and include restlessness, drop in milk production, standing to be mounted, presence of clear mucus at, swelling and reddening of vulva. Oestrus lasts for an average of 18 hours, ranging from 12 to 24 hours in cows (Hafez and Hafez, 2000; Frandson et al., 2009), while spontaneous ovulation brought about by a decrease in FSH levels in the blood and an increase in luteinizing hormone levels occur 10 to 14 hours after the end of oestrus (Frandson et al., 2009). The met-oestrus period is characterized by the formation of the corpus luteum which attains maximum size and becomes fully functional producing progesterone during the di-oestrus period (Frandson et al., 2009). The progesterone produced University of Ghana http://ugspace.ug.edu.gh 5 is responsible for the maintenance of pregnancy if, pregnancy ensues. At the end of di- oestrus, luteolysis (regression) of the corpus luteum begins under the influence of prostaglandin F2α secreted from the uterine endometrium, if pregnancy does not result. 2.2 POSTPARTUM ANOESTRUS IN CATTLE Postpartum anoestrus is the period after calving and before normal ovarian cycles are re- established (McDougall, 1994). It is marked by absence of ovulation and expression of oestrus. According to Stagg et al. (1995), although there is follicular development during this period, they do not mature to ovulate. The postpartum period plays a major role in reproductive efficiency in cattle (Short et al, 1990). Prolonged postpartum anoestrous periods tend to extend calving intervals thus preventing the attainment of a desirable calving interval of about 365 days (one year) for efficient cattle production (Payne, 1990; Montiel and Ahuja, 2005). Consequently, this causes economic loss to farmers. Obese et al. (2009) in a review indicated that prolonged postpartum anoestrus is a major infertility problem militating against the achievement of a desirable calving interval of 12 months in indigenous breeds of cows managed in extensive pasture-based systems in Ghana. Prolonged postpartum anoestrous periods ranging from 101 to121 days, and extended calving intervals ranging from 413 to 455 days have been reported for the N’dama, West African Shorthorn and Sanga cows in Ghana (Osei et al., 1993, Karikari et al., 1995; Obese et al. 1999). Ideally, to achieve a calving interval of 365 days, a postpartum cow has to resume normal ovarian cyclicity (ovulation and oestrus), be inseminated, and conceive within 85 days of calving (Stagg et al., 1995, Mihm, 1999). Follicular growth generally resumes within 7–10 days in a majority of cows associated with a transient FSH rise that occurs within 3–5 days of parturition. Delayed or lack of ovulation has been attributed to reduced LH pulsatility (Yavas and Walton, 2000; Crowe, 2008). The University of Ghana http://ugspace.ug.edu.gh 6 inhibition of LH pulse frequency and suppression of blood concentrations of glucose, insulin and IGF-I leads to low oestradiol concentration, preventing the induction of gonadotrophin surge necessary for ovulation to occur in cattle (Diskin et al., 2003; Peter et al., 2009; Scaramuzzi et al., 2011). A low LH pulse frequency in anoestrous cows is attributed to inhibition of pulsatile release of gonadotrophin-releasing hormone (GnRH) from the hypothalamus (Roche and Diskin, 2001; Wettemann et al., 2003). The release of GnRH and LH is modulated by ovarian steroids (oestrogen and progesterone) and by extra-ovarian factors including level of nutrition, body condition score and suckling (Imakawa et al., 1986; Williams et al., 1996). 2.3 FACTORS AFFECTING POSTPARTUM ANOESTROUS INTERVAL The duration of postpartum anoestrus is influenced by several factors including level of nutrition, body condition, suckling, season of calving, breed, age or parity of cow, milk yield, and peri-parturient disease (ketosis, mastitis, retained placenta, metritis). Among these factors, nutrition and suckling are most important (Mukasa-Mugerwa, 1989; Short et al., 1990; Hafez and Hafez, 2000; Wettemann et al., 2003), while the other factors may modulate the effects provoked by nutrition and suckling. 2.3.1 Effect of nutrition on postpartum anoestrous interval Inadequate nutrition relative to metabolic demands is a major factor contributing to prolonged postpartum anoestrous, particularly among cows dependent upon natural forages for most or all of their feed requirements (Jolly et al., 1995). In cattle, follicular growth, maturation and ovulatory capacity is influenced by nutritional status (Diskin et al., 2003). In beef and dairy cattle, nutrition before or after calving plays a major role in the timing of the onset of University of Ghana http://ugspace.ug.edu.gh 7 oestrous cyclicity after calving, the normality of its expression and conception rate (Robinson et al., 2006). According to Diskin et al. (2003), and Drackley and Cardoso (2014), negative energy balance or reduced dietary intake in the early postpartum period adversely affects the size and ovulatory fate of the dominant follicle and prolongs postpartum anoestrus in cows. The inadequate intake of nutrients (limited dietary energy and protein intake) relative to metabolic demands, especially in the dry season when forage availability is scarce and its quality poor, has contributed to prolonged postpartum anoestrus in indigenous cows in smallholder dairy systems in Ghana depending mainly on natural forages for most or all of their feed requirements (Obese et al., 2009). Poor nutritional status reduces systemic concentrations of LH, IGF-I, and oestradiol, resulting in delay in occurrence or decrease in the magnitude of the ovulatory surge of LH induced by oestradiol, leading to anovulation (Short et al., 1990; Jolly et al., 1995). As a management strategy, improving the nutrition of cows by strategic supplementation of poor quality forages with crop residues, agro-industrial by-products, leguminous browse plants and multi-nutrient feed blocks (urea-molasses) especially during the dry season should help in improving the productive and reproductive performance of cattle in smallholder dairy systems in Ghana. These supplements which are energy and/or protein sources increase the energy and/or protein supply to the host animal by alleviating deficiencies in microbial fermentation, improving the rumen environment and increasing production of microbial proteins (Leng, 1992; Osuji, 1994). Body condition score (BCS) is a subjective visual and tactile measure of body condition. Temporary changes in BCS are used to monitor nutritional and health status of cows during their productive cycle (Berry et al., 2007). BCS has been correlated with reproductive performance in cows (Berry et al., 2003; Buckley et al., 2003), and supports the premise that nutritional status affects reproductive function. The BCS at key periods in lactation, as well University of Ghana http://ugspace.ug.edu.gh 8 as BCS changes have been associated with the resumption of oestrus cycles and reproductive success in cows (Pryce et al., 2001). Cows in low body condition at calving, or cows that suffer excess BCS loss early postpartum, are less likely to ovulate, and have increased calving to conception intervals (Berry et al., 2007; Roche et al., 2009) due to impaired oocyte competence (Snijders et al., 2000). According to Stagg et al. (1998), cows that calve down in poor BCS (<2.5 on a five point score) are more likely to have a prolonged anoestrous period due to lower LH pulse frequency. It has been recommended that cows have a BCS of 2.75- 3.0 (on a scale of 5) at calving, and that they be managed to lose BCS of not more than 0.5 units between calving and first service (Crowe, 2008). 2.3.2 Effect of suckling on postpartum anoestrous interval Suckling is a factor that has a dramatic effect on the postpartum anoestrous interval. Nursing a calf suppresses postpartum ovarian activity in both B. taurus and B. indicus cattle (Oxenreider and Wagner, 1971; Williams, 1990). Suckling causes suppression of GnRH secretion, leading to insufficient pulsatile LH release necessary for growth, maturation and ovulation of ovarian follicles. Ovulation is delayed, thus extending the postpartum anoestrous period (Williams et al., 1996). The maternal bond between the cow and her own calf is also a major factor resulting in delayed resumption of ovulation, and is dependent upon visual and/or olfactory signals between dam and calf (Macmillan, 1983). Furthermore, frequency of suckling may play a role in the inhibitory effect of suckling on the postpartum anoestrous interval. For example, suckling twice or three times a day was found to increase the duration of postpartum anoestrus and decreased LH concentration compared with suckling once a day (Short et al., 1990). Also, Crowe (2008) reported that cows that were allowed to suckle their calves once University of Ghana http://ugspace.ug.edu.gh 9 per day had reduced postpartum anoestrous intervals compared with ad libitum suckled control cows (51 versus 79 days). Furthermore, N’dama cows that suckled their calves for less than 3 months had shorter postpartum anoestrous intervals compared with those that suckled longer than 3 months (85 ± 48.8 versus 256.4 ± 135.5 days; Osei et al., 1997) in the humid forest zone of Ghana. Thus regulation of the suckling stimulus may be a viable management option to decrease postpartum anoestrous interval. Postpartum anoestrous intervals can be decreased by early weaning or partial weaning (restricting suckling to short period of time each day). Such weaning treatments have been reported to increase LH pulse frequency, stimulate ovulation and reduce calving intervals in tropical cows (Mukasa- Mugerwa et al., 1991; Tegegne et al., 1992). Early weaned calves or those on restricted suckling would however need extra nutritional supplements to ensure continued growth (Mukasa-Mugerwa et al., 1991). University of Ghana http://ugspace.ug.edu.gh 10 2.3.3 Effect of season on postpartum anoestrous interval Seasonal effects on the postpartum anoestrous period include the direct effects of photoperiod (Garel et al., 1987; Savio et al., 1990) and indirect effects of seasonal differences in nutrient quality and quantity. Postpartum anoestrous intervals have been found to be shorter when calving occurred at a time when the photoperiod was increasing (McDougall et al., 1995a). For example, cows calving from late spring to early fall had shorter postpartum anoestrous intervals than cows calving from late fall to early spring (King and MacLeod, 1983; Smeaton et al., 1986). The hormone melatonin is known to influence the frequency of GnRH release from the hypothalamus (Mwaanga and Janowski, 2000). It is believed that during shorter day-length there is high release of melatonin which tends to decrease the frequency of GnRH release, extending the anoestrous period. Also, increasing day length has been reported to increase circulating concentrations of IGF-I which may lead to reduction in the length of postpartum anoestrous interval (Dahl et al., 1997; Dahl et al., 2000). The postpartum anoestrous interval in a mixed herd of N’dama and WASH on smallholder farms in Kumasi was reduced when cows calved in the dry season compared with those calving in the wet season (105 ±18.8 versus 119 ± 21.9 days; Osei et al., 1993). Also, Okantah et al. (2005) working with Sanga cows on the Accra Plains reported shorter anoestrous intervals in cows that calved in the dry season than those that calved in the wet season (94.1 ± 8.8 versus 108.6 ± 12.0 days). This observation may be due to the fact that cows calving towards the end of the dry season take advantage of improved nutritional conditions during the subsequent rainy season to meet their requirements for maintenance, growth and lactation, and this promotes earlier resumption of ovarian cycles (Karikari et al., 1990; Abeygunawardena and Dematawewa, 2004). University of Ghana http://ugspace.ug.edu.gh 11 2.3.4 Effect of breed and genotype on postpartum anoestrous interval Mwaanga and Janoski (2000) in a review have indicated that suckled dairy cows have longer postpartum intervals than suckled beef cows, while milked dairy cows have shorter postpartum intervals than suckled beef cows. Also, some studies have shown that dairy genotypes have longer postpartum anoestrous intervals than beef genotypes, and these effects are more pronounced at first parity and at lower dietary intakes (Hansen et al., 1982; Short et al, 1990). Differences also have been reported between beef breeds, with the effect being more pronounced at lower dietary intakes (Dunn et al., 1969; Bellows and Short, 1978). The effect of genotype on postpartum anoestrus may be due to physiological differences among breeds, amount of milk produced and feed intake (Short et al, 1990). The effect of breed on postpartum anoestrous interval is an important factor, and even though breed usually is predetermined, this effect must be considered when managing postpartum cows. 2.3.5 Effect of Age and parity on postpartum anoestrous interval Younger cows tend to have longer postpartum anoestrous intervals than older cows (Short et al., 1990; McDougall et al., 1995b; McDougall et al., 1998) and this has been attributed to the additional energy requirement of young cows for growth as well as milk production. In Zebu cattle, postpartum anoestrous interval was extended in first-calf heifers and older cows and was shortest in cows of intermediate ages of between 6 to 9 years old (Mukasa-Mugerwa, 1989). Primiparous cows have been observed to have longer intervals to first ovulation than multiparous ones, and cows with lower energy balances have longer intervals than those with higher energy balances (Britt et al., 1993). For example, Tanaka et al. (2008) recorded more days to first ovulation (31.8 ± 8.3 days) in primiparous than multiparous cows (17.3 ± 6.3 days). Lucy (2001) University of Ghana http://ugspace.ug.edu.gh 12 indicated that primiparous cows have energetic demands for growth as well as lactation and may be in greater negative energy balance than multiparous cows. Age and dystocia are associated with body weight and could increase postpartum anoestrous interval and therefore delay rebreeding (Brinks et al., 1973; Doornbos et al., 1984). Oestrus does not occur in some breeds of cattle until a certain body weight is attained (Short et al., 1990). In situations where puberty occurs at an early age and the cow is served, parturition difficulties such as large calf size may arise and this in turn will affect the resumption of ovarian cycles after calving (Mwaanga and Janowski, 2000). The adverse effects of dystocia can be overcome at least partially by providing early assistance (Bellows et al., 1988). 2.3.6 Effect of peri-paturient diseases on postpartum anoestrous interval According to Crowe et al. (2014), postpartum uterine infection is a major contributor to reduced fertility in cattle through the disruption of ovarian function. Following parturition, the uterus becomes contaminated with bacteria, and although many animals can clear this contamination, infection persists in up to 20% of cows as endometritis (Sheldon et al., 2009). Bacterial products such as the endotoxin, lipopolysaccharide or immune mediators produced in response to infection suppress pituitary LH secretion and are associated with inhibition of folliculogenesis, cystic ovarian follicles, and decreased secretion of oestradiol by ovarian follicles. This consequently prolong the interval from calving to ovulation, and also decrease conception rate (Opsomer et al., 2000; LeBlanc et al., 2002; Williams et al., 2007; Sheldon et al., 2009). It has been observed that retained foetal membranes delay uterine involution, predisposing cows to endometritis or metritis (Mellado and Reyes, 1994). University of Ghana http://ugspace.ug.edu.gh 13 Lameness has also been associated with delayed oestrus in dairy cows when it occurs peripartum (Peeler et al., 1994). Lame cows may be physically unable to exhibit oestrus, or they may secrete endogenous opioid peptides thereby inhibiting LH pulse frequency (Peeler et al., 1994). Cows with subclinical mastitis show pronounced reduction in preovulatory oestradiol and androstenedione concentrations output, which leads to low and delayed LH surge. This affects follicular growth and development leading to delayed ovulation (Lavon et al., 2011). 2.3.7 Effect of milk yield on postpartum anoestrous interval The effect of milk yield on postpartum anoestrous interval is more pronounced in high yielding dairy cows, where milking frequency (three times per day) is higher than the average for low producing cows. Frequent milking may have a negative effect similar to that of a suckling calf on the dam’s reproductive system, by blocking LH secretion (Mwaanga and Janowski, 2000). High milk yield negatively affects reproductive performance in dairy cows (Fonseca et al., 1983). This is due to negative energy balance caused by the inability of dietary intake of the cow to meet the energy requirements for milk production and reproductive activities, especially during early lactation. Milk production gets preference over reproduction, therefore prolonging the postpartum anoestrous interval (Butler, 2003; 2005). University of Ghana http://ugspace.ug.edu.gh 14 2.4 METABOLIC HORMONES AND NUTRITIONAL METABOLITES ASSOCIATED WITH OVARIAN FUNCTION IN CATTLE A number of metabolic hormones and nutritional metabolites in the blood play important roles in reproductive function, especially the resumption of ovarian function in cows during the postpartum period (Wettemann et al., 2003; Konigsson et al., 2008). The metabolic hormones include GH, insulin, IGF-I and leptin. The nutritional metabolites include NEFA, BHB, glucose, cholesterol, total protein, albumin, globulin, creatinine and urea. According to Lindsay et al. (1993), changes in circulating concentrations of metabolic hormones and nutritional metabolites are important signals of the metabolic state of the animal and ovarian function. 2.4.1 Role of growth hormone (GH) in ovarian function in cattle GH is secreted by the somatotroph cells within the anterior pituitary gland and stimulates growth in animals. It has many metabolic effects on carbohydrate, fat, and protein metabolism (Lucy, 2001). In peri-parturient cows, GH orchestrates numerous changes in various tissues in support of lactation including reduction in insulin sensitivity, an increase in hepatic gluconeogenesis, and reduced whole body utilization (Bell and Baumans, 1997). Hence, the early lactation period is characterized by reduced plasma insulin and IGF-I and elevated GH and NEFA concentrations, according to Bossis et al. (1999). In nutritionally induced anoestrous heifers, mean serum concentration and pulse amplitude of GH were both increased during the last two oestrous cycles before onset of anoestrus, while pulse frequency remained unchanged. In cattle, the main effects of GH on reproduction appear to be operated through its regulatory effects on hepatic IGF-I synthesis and secretion, with no evidence to-date of GH-dependent follicular IGF-I synthesis or a University of Ghana http://ugspace.ug.edu.gh 15 direct effect of GH on bovine follicles (Lucy et al., 1999; Gong, 2002). This led Lucy et al. (1999) to conclude that GH has a facilitatory rather than an obligatory role in reproduction. 2.4.2 Role of insulin-like growth factor-I (IGF-I) in ovarian function in cattle IGF-I is a single chain polypeptide which plays an important role in regulation of cell growth and differentiation (Jones and Clemmons, 1995; Cohick, 1998; Baumrucker and Erondu, 2000). There is release of IGF-I from the liver under the influence of GH, as well as local tissue synthesis that can affect postnatal growth and reproduction (Le Roith et al., 2001). IGF-I is a potential mediator of nutritional effects on reproduction (O’Callaghan and Boland, 1999; Zulu et al., 2002; Velazquez et al., 2008). Nutrient intake influences the levels of circulating IGF-I in cattle. High energy and/or protein intake increased circulating IGF-I concentrations, while levels were reduced by lower protein or energy intake (Cohick, 1998). According to Patton et al. (2007), higher serum IGF-I concentrations in cows lead to earlier commencement of luteal activity and greater conception rate to first service. In numerous studies, cows that resumed ovarian function early had higher circulating concentrations of IGF-I than late cycling or non-cycling cows, reflecting the importance of IGF-I in the early postpartum period for the subsequent resumption of ovarian cyclicity. This has been demonstrated in beef as well as dairy cows (Beam and Butler, 1997; Roberts et al., 1997; Taylor et al., 2003; Kawashima et al., 2007; Tamadon et al., 2011; Obese et al., 2012; Samadi et al., 2013). For example, Obese et al. (2012) reported that early-cycling (23.2 ± 1.26 ng/mL) or late-cycling (19.5 ± 1.38 ng/mL) Sanga cows had higher plasma concentrations of IGF-I than non-cycling cows (14.7 ± 1.38 ng/mL) when they grazed extensively on natural pasture in the Accra Plains. Also, Saleh et al. University of Ghana http://ugspace.ug.edu.gh 16 (2011) recorded higher IGF-I concentrations in cycling than non-cycling cows (53.16 ± 1.41 versus 38.46 ± 1.58 ng/mL) in indigenous Egyptian cows. Circulating concentrations of IGF-I stimulates ovarian function by acting synergistically with gonadotrophins to promote follicular growth, differentiation and steroidogenesis of the ovarian cells (Lucy, 2000; Chagas et al., 2007; Scaramuzzi et al., 2011; Crowe et al., 2014). Low circulating concentrations of IGF-I may thus contribute to reduced follicular responsiveness to a given level of gonadotrophic support, low oestradiol synthesis, and anovulation in postpartum cows (Beam and Butler, 1999; Peter et al., 2009). 2.4.3 Role of insulin in ovarian function in cattle Insulin is secreted from the pancreatic β-cells (Butler, 2014). It is involved in glucose homeostasis and also serves as a metabolic signal influencing pituitary release of LH (Monget and Martin, 1997), and also regulates ovarian responsiveness to gonadotrophins (Stewart et al., 1995; Diskin et al., 2003). Lower levels of insulin may thus, suppress LH release and consequently delay ovulation. For example, Sinclair et al. (2002) demonstrated that postpartum anoestrous beef cows with lower plasma concentrations of insulin were unable to ovulate a dominant follicle in response to restricted suckling, unlike cows with higher plasma concentrations of insulin, notwithstanding an increase in LH pulse frequency. Also, during the postpartum period, dairy cows fed total mixed ration diets designed to increase circulating insulin concentrations resumed ovarian cyclicity earlier and had better conception rate to first service (Gong et al., 2002). University of Ghana http://ugspace.ug.edu.gh 17 Insulin is required for increased synthesis of IGF-I in the liver in response to elevated concentrations of GH in order to increase oestradiol production by the dominant follicle and also increase LH receptors for ovulation and corpus luteum development (Lucy, 2000; Garnsworthy et al., 2008). Plasma concentrations of insulin were reported to be higher in early than in late- cycling (4.16 ± 0.24 versus 4.00 ± 0.26 µU/mL) grazing Sanga cows, during weeks 1 to 10 in the postpartum period (Damptey, 2012). According to Drackley and Cardoso (2014), insulin concentrations generally reflect energy status and dietary adequacy, and may be a primary link between the metabolic and reproductive systems. Dietary restriction and negative energy balance are factors that reduce circulating concentrations of insulin (Mackey et al., 2000; Sinclair et al., 2002). Cummins et al. (2012) reported reduced insulin concentrations in Holstein cows in pasture-based system during lactation-induced negative energy balance. 2.4.4 Role of leptin in ovarian function in cattle Leptin is a protein hormone produced in adipose tissues (Zhang et al., 1994; Dyer et al., 1997). It acts directly on the hypothalamus to regulate food intake and the whole-body energy balance, and has a direct action at the level of the ovary (Spicer, 2003). Leptin is a metabolic hormone that regulates nutritional effects on reproductive function and is a possible signal between feed intake and secretion of LH (Clarke and Henry, 1999; Williams et al., 2002). Kadokawa et al. (2000) working with Holstein cows found a positive relationship between leptin concentration and onset of luteal function in cows. According to Wettemann et al. (2003), leptin may have a University of Ghana http://ugspace.ug.edu.gh 18 direct effect on the pituitary to increase secretion of LH, and on the ovary to regulate steriodogenesis. Chronic and acute changes in nutrition affect systemic leptin concentrations in sheep (Blache et al., 2000) and cattle (Ehrhardt et al., 2000). In dairy cows, elimination of the energy deficit in early lactation was associated with a doubling of plasma concentrations of leptin (Block et al., 2001). Damptey (2012), however working with Sanga cows on the Accra Plains reported that there was no significant influence of leptin concentrations on resumption of their ovarian activity. The plasma leptin concentrations recorded in that study were similar in early-cycling (1.16 ± 0.02 ng/mL), late cycling (1.13 ± 0. 02 ng/mL), and non-cycling cows (1.09 ± 0.02 ng/mL). 2.4.5 Role of glucose in ovarian function in cattle Glucose supplies energy for life processes in the body of an animal, and is the primary metabolic fuel used by the central nervous system (Saleh et al., 2011; Butler, 2014). Insufficient nutrient intake can reduce circulating glucose concentration (Ndlovu et al., 2007). Glucose is a metabolic signal providing information for the control of GnRH secretion, as glucose availability influences both tonic and surge modes of LH secretion, through its effects on GnRH (Diskin et al., 2003; Butler, 2014). Inadequate availability of utilizable glucose reduces hypothalamic release of GnRH leading to a decrease in LH release, and eventually delaying or preventing ovulation (Hess et al., 2005). Damptey et al. (2014) reported that glucose concentrations were similar in early cycling (3.61 ± 0.05 mmol/L), late cycling (3.59 ± 0.06 mmol/L) and non-cycling (3.59 ± 0.06 mmol/L) Sanga cows, between weeks 1 to 13 postpartum when they grazed on natural pasture on the Accra Plains. In a study using indigenous Egyptian cows, Saleh et al. University of Ghana http://ugspace.ug.edu.gh 19 (2011) reported that serum concentration of glucose was higher in cycling than non-cycling cows (6.11 + 0.29 mmol/L versus 4.08 + 0.1 mmol/L). The normal physiological levels of glucose in cattle range from 2.2 to 5.6 mmol/L (The Merck Veterinary Manual, 2010). 2.4.6 The role of non-esterified fatty acids (NEFAs) in ovarian function in cattle During early lactation, most cows cannot consume sufficient energy-yielding nutrients from voluntary dry matter intake after calving to meet energetic requirements for milk production. They therefore experience negative energy balance (Drackley and Cardoso 2014). During this period, there is high body tissue mobilization yielding high circulating concentrations of NEFA and ketones (Wathes et al., 2007; Butler, 2014; Esposito et al., 2014). NEFA can be partially metabolized to ketone bodies such as BHB and distributed to other tissues for energy metabolism, or they may be used to synthesize fats in the liver (Butler, 2014). Excessively high NEFA concentrations due to negative energy balance result in fatty infiltration of the liver and is associated with a higher incidence of peri-parturient metabolic diseases (Cameron et al., 1998; Jorritsma et al., 2003; Walsh et al., 2007; McArt et al., 2012). Furthermore, elevated concentrations of NEFA and BHB have been linked to prolonged postpartum anoestrous interval in cows (Reist et al., 2000; Giuliodori, et al., 2011). According to Giuliodori et al. (2011), high NEFA and GH concentrations could antagonize insulin action and create a state of insulin resistance decreasing the sensitivity of the ovary to LH and FSH, leading to a delay in the resumption of ovulation postpartum. University of Ghana http://ugspace.ug.edu.gh 20 2.4.7 Role of cholesterol in ovarian function in cattle Cholesterol is a major component of blood plasma and an essential constituent of cell membranes. It is also a precursor for steroid hormones and it plays important role in the manufacture of bile acids, steroids, hormones and vitamin D (Edfers-Lilja et al., 1980). Macial et al. (2001) reported that decrease in plasma cholesterol concentration led to a reduction of plasma concentrations of IGF-I and progesterone which resulted in the suppression of luteal function and eventually delayed ovulation in dairy cows. Plasma cholesterol concentrations were reported to be similar in early-cycling (2.44 + 0.11mmol/L), late-cycling (2.49 + 0.12mmol/L) and non- cycling (2.47 + 0.12mmol/L) Sanga cows grazed extensively on natural pasture (Damptey et al., 2014) during week 1 to 13 postpartum. However, Saleh et al., (2011) recorded high serum concentration of total cholesterol in cyclic than non-cyclic indigenous Egyptians cows (4.52 ± 0.09 mmol/L versus 4.19 ± 0.03 mmol/L). The nutritional status and breed of cows may account for these differences. Cholesterol levels in the blood is influenced by diet, age and sex of the animal (Abou-Tarboush and Dawood, 1993). The normal total cholesterol concentration for cows have been reported to range from 1.6 to 5.0 mmol/L (The Merck Veterinary Manual, 2010). 2.4.8 Role of proteins in ovarian function in cattle The major blood proteins include albumin, globulin and fibrinogen. Total protein and albumin levels reflect availability of protein, and their concentrations decline as a result of protein deficiency (Ndlovu et al., 2007). Blood proteins assist in the regulation of cellular activity and functioning, and act as circulatory transport molecule for lipids, hormones, vitamins, enzymes and metals (Quintavalla et al., 2001). Total protein levels are lower in younger animals and University of Ghana http://ugspace.ug.edu.gh 21 higher in mature animals, whilst albumin levels are lower at birth and then increase with age (Doornenbal et al., 1988; Otto et al., 2000). Albumin is the main protein of blood plasma. It plays a role in regulating the colloidal osmotic pressure of blood. It also functions primarily as a carrier protein for steroids, fatty acids, and thyroid hormones, and plays a role in stabilizing extracellular fluid volume. Albumin in serum is very sensitive and serves as an early nutritional indicator of protein status (Agenas et al., 2006). Albumin concentration is affected by age (Otto et al., 2000), malnutrition (Ndlovu et al., 2007) and level of infection. Infections in animals can result in considerable decrease in serum albumin concentrations compared to uninfected animals (Orhue et al., 2005). Circulating globulin concentrations give indication of an animal’s immune state and its ability to fight diseases and infections (Kapele et al., 2008; Shrikhande et al., 2008). The normal total protein, albumin and globulin concentration in the blood of cattle range between 67-85g/L, 25-38 g/L and 30-35 g/L respectively (Otto et al., 2000; The Merck Veterinary Manual, 2010). Damptey et al. (2014) reported that plasma total protein, albumin and globulin concentrations did not influence the resumption of ovarian function in Sanga cows grazing extensively on natural pasture on the Accra Plains. The concentrations of these three nutritional metabolites were similar in early-cycling, non- cycling and late cycling cows Sanga cows, according to the authors. University of Ghana http://ugspace.ug.edu.gh 22 2.4.9 Role of blood urea nitrogen in ovarian function in cattle Urea is the principal end-product of catabolism of proteins (Coles, 1996). The levels of urea nitrogen in blood is a good indicator of concentrations of rumen ammonia, and this is related closely to intake and solubility of dietary nitrogen-containing compounds (Greenwood et al., 2002). Insufficient availability of dietary energy is known to increase rumen ammonia and blood urea concentrations by limiting microbial synthesis of protein (Murphy, 1999). Also, during periods of energy restriction, the shortfall in energy may be met by catabolism of body proteins, which results in increased urea concentrations in the blood (Greenwood et al., 2002). Damptey et al. (2014) recorded lower plasma urea concentrations in early-cycling (5.99 ± 0.16 mmol/L) than in late-cycling (6.57 ± 0.17 mmol/L) and non-cycling (6.59 ± 0.17 mmol/L) Sanga cows in an extensive pasture-based system on the Accra Plains. Ahmad et al. (2004) recorded similar mean blood urea concentrations of 1.71 ± 0.13 mmol/L and 1.86 ± 0.19 mmol/L for cyclic and non-cyclic crossbred cows respectively in Pakistan. Normal urea nitrogen concentration for cows range from 3.6-8.9 mmol/L (The Merck Veterinary Manual, 2010). 2.4.10 Role of creatinine in ovarian function in cattle Creatinine is a by-product of the breakdown of creatine and phosphocreatine in the muscle (Miller et al., 2004; Ndlovu et al., 2007). Creatinine along with blood urea nitrogen, are excellent indicators of protein metabolism and kidney function (Quintavalla et al., 2001). Blood creatinine concentrations vary due to an animal’s diet, breed, muscle mass and sex (Otto et al., 2000; Miller et al., 2004). Reduced concentrations of creatinine indicate prolonged active tissue protein catabolism (Agenas et al., 2006). Creatinine is directly related to muscle mass because it University of Ghana http://ugspace.ug.edu.gh 23 is a product of muscle metabolism, and as a result, it is significantly correlated to live weight (Whittet et al., 2004). The normal range of creatinine in the blood for cattle is 44 to 194 mmol/L (The Merck Veterinary Manual, 2010). Damptey et al. (2014) reported higher plasma concentrations of creatinine in early-cycling (101.81 ± 1.82 mmol/L) than in late-cycling (94.03 ± 1.99 mmol/L) or non-cycling cows (97.40 ± 97.40 mmol/L). Sanga cows which grazed extensively on natural pasture in the Accra Plains. University of Ghana http://ugspace.ug.edu.gh 24 CHAPTER TREE MATERIALS AND METHODS 3.1 Location of study The study was conducted at the Animal Research Institute’s Katamanso Station located on latitude 05º 44' N and longitude 00º 08' W, on the Accra Plains of Ghana. The area has a bimodal rainfall pattern with the major wet season occurring from April to July, and the minor season from September to November. The remaining months constitute the dry period. The annual rainfall and temperatures range from 600 to 1000 mm and 20 ºC to 34 ºC, respectively. 3.2 Management of Animals Multiparous Friesian-Sanga cows (n=16; Plate 3.1) calving between April and May, 2014 were used in the study. At the start of the experiment, the cows recorded a mean (± SEM) body weight (BW) of 295.2 ± 10.5 kg; and BCS 5.1 ± 0.2 on a scale of 1 to 9 (Nicholson and Butterworth, 1986). The animals were housed in an open kraal and allowed to graze from 05:00 to 10:00 h and 13:00 to 16:00 h daily, mainly on natural pastures comprising mainly Panicum maximum, Sporobolus pyramidalis and Vertiveria fulvibarbis as dominant grass species, and Griffonia simplicifolia, Bafia nitida and Milletia throningii as the main browse species in the grazing area. The cows were not given any feed supplements. Water was provided twice daily (morning and evening). Cows were milked twice a day during the rainy season and once a day during the dry season. Partial milking was practised; calves were separated from their dams in the evening and brought in to suckle for a few minutes to stimulate milk let-down before milking. Milk was collected from two quarters of the udder and the other two quarters were reserved for the calves. Natural mating was practised with bulls running freely with females all year round, and the University of Ghana http://ugspace.ug.edu.gh 25 calves were weaned at 6 months of age. Cows and their calves were weighed monthly and BCS of cows determined weekly, using a 9-point scale (1 = very thin and 9 = obese) (Nicholson and Butterworth, 1986). The animals were treated against ecto-parasites, mainly ticks, fleas and mange mites using a pour-on acaricide (Flumethrin 1 % m/v). For endo-parasites, the animals were treated with the antihelminth, Albendazole (10 %) once per month during the dry season and fortnightly in the wet season. They were treated against diseases as the need arose and vaccinated against Contagious Bovine Pleuropneumonia once a year. Cows were monitored for oestrus by means of visual observations on the pasture at least two times per day (06:00 h and 18:00 h). The cycling or non-cycling status of cows was based on oestrus detection and confirmed using progesterone profiles. Plate 3.1: Friesian-Sanga crossbred cow University of Ghana http://ugspace.ug.edu.gh 26 3.3 Blood sampling for progesterone, metabolic hormones and nutritional metabolites Blood samples were collected from cows once every week, from week 1 to 16 postpartum at 08:00 h by jugular venipuncture into a 10 mL heparinised vacutainer tube (BD Vacutainer Systems, Plymouth, UK) for analysis of progesterone, metabolic hormones (GH, IGF-I, insulin), and nutritional metabolites (cholesterol, total protein, albumin, urea and creatinine). Blood samples for determining concentrations of glucose were collected into evacuated tubes containing fluoride oxalate. All samples collected were then placed on ice immediately after collection, and plasma was separated by centrifugation at 1800×g for 15 min at 4 °C. Plasma was stored at -20 °C, until assayed for the metabolic hormones and nutritional metabolites. 3.4 Measurement of progesterone, metabolic hormones and nutritional metabolite concentrations The progesterone assays were carried out at the Public Health Reference Laboratory of the Korle Bu Teaching Hospital, Accra, Ghana; while assays for GH, IGF-I and insulin were carried out at the Physiology Laboratory of the School of Animal Biology, University of Western Australia, Australia. The concentrations of the nutritional metabolites were measured at the Central Laboratories, Korle Bu Teaching Hospital, Accra, Ghana. The concentrations of GH, IGF-I and insulin in plasma samples were measured from week 1 to 10 postpartum, while the concentrations of the nutritional metabolites in the plasma samples were measured at weeks 1, 3, 5 and 9 postpartum. University of Ghana http://ugspace.ug.edu.gh 27 3.4.1 Progesterone Assay Resumption of postpartum ovarian activity was determined by measuring the progesterone concentrations in plasma samples from cows. Plasma progesterone concentrations were measured using a commercial Progesterone ELISA kit (NovaTec Immundiagnostica GmbH, Dietzenbach, Germany), following the recommendation of the manufacturer. Cows were classified as having resumed ovarian activity when plasma progesterone concentration of ≥1 ng/mL was recorded in two consecutive weekly samples (Tamadon et al., 2011). Based on the resumption of ovarian activity, cows were classified as early-cycling (resumed activity ≤ 56 days postpartum), late-cycling (resumed ovarian activity 57-112 days postpartum) or non-cycling (no resumption by 112 days postpartum). The ELISA was validated for determination of progesterone in cattle plasma by including standard samples for cattle. In the assay, 50 µL standards (17α OH Progesterone) with concentrations (0, 0.2, 0.4, 1.6, 6.4, 19.2, ng/mL), control, and test samples were dispensed into their respective ELISA wells (coated with anti 17α OH Progesterone antibodies) in duplicates; 50 µL 17 α OH Progesterone-HRP conjugate was then added to each well. The wells were covered with a foil supplied in the kit and incubated for 1 hour at 37oC. After incubation the foils were removed, content of the wells were aspirated and washed twice with 300 µL of distilled water. Next, 100 µL TMB (Tetra methyl benzidine) substrate solution was then dispensed into all wells and incubated for exactly 15 min, at room temperature in the dark. After this, 100 µL stop solution (Sulphuric acid) was dispensed into wells in the same order and at the same rate as done for the TMB Substrate solution. The microplate was shaken gently. Any blue colour developed during the incubation turned into yellow. University of Ghana http://ugspace.ug.edu.gh 28 The absorbance of the standards and test samples were determined using the Original Multiskan Ex instrument (Thermo Electron Corporation, USA) at a wavelength of 450 nm within 30 minutes of termination of the kinetic reaction. A dose response curve was used to interpolate the concentration of progesterone in the various samples. This was done by plotting the absorbance of each duplicate sample of the standards (on the Y-axis) versus their corresponding progesterone concentration in ng/mL (on the X-axis) on a linear graph paper, to obtain a standard curve. The average absorbance (of the duplicate) of the test samples were located on the X-axis and their corresponding progesterone concentrations were read from the Y-axis on the graph. The progesterone ELISA assay had a sensitivity of 0.09 ng/mL. University of Ghana http://ugspace.ug.edu.gh 29 3.4.2 Growth hormone (GH) Assay The concentration of GH in plasma samples was determined following the radio-immunoassay technique (RIA) of Downing et al. (1995) in a single assay. The assay sensitivity was 0.03 ng/mL. The intra-assay coefficients of variation were 7.4%, 6.8% and 5.3% for low, medium and high concentrations, respectively. The assay included 6 replicates each of three quality control pools. On Day 1, plasma samples (100 µl), standards in 0.05M phosphate buffer + 0.25% bovine serum albumin (BSA) (100 µL) were diluted to 400 µL with 0.05M phosphate buffer; and 50 µL of antiserum was added and incubated at 4°C overnight. On Day 2, 50 µl of tracer was added, vortexed and incubated for a further 48 hours, after which time donkey anti-rabbit serum (50 µl: 1:7 in 0.05M phosphate buffer was added to all tubes (except Total controls), before centrifugation at 1500 g for 25 minutes at 4°C. The supernatant was decanted and the activity of the precipitate was determined on a gamma counter (Plate 3.2). University of Ghana http://ugspace.ug.edu.gh 30 Plate 3.2: Packard Cobra-II Auto Gamma Counter (Source: Physiology Laboratory, University of Western Australia) 3.4.3 IGF-I Assay Plasma concentrations of IGF-I were measured in duplicate by the chloramine-T RIA method described by Gluckman et al. (1983). Interference by binding proteins was minimized by acid- ethanol cryo-precipitation method validated for ruminants by Breier et al. (1991). The assay sensitivity was 0.05 ng/mL and the intra and inter-assay coefficients of variation (CV) were 6.3% and 7.9% respectively. University of Ghana http://ugspace.ug.edu.gh 31 On Day 1 of the assay, 100 µL of plasma and quality controls were extracted by mixing with 400 µL of acid-ethanol (1:7 HCL: absolute ethanol) in glass tubes. The tubes were then vortexed and left to stand at 20˚C for 30 min, before centrifugation at 1,500 x g for 30 mins at 4˚C. The supernatant was then decanted into plastic assay tubes and 0.855N Tris (base) was added to neutralise the solution (e.g, 250 µL supernatant + 60 µL Tris) before it was left to stand overnight at -20˚C. On Day 2, the samples were centrifuged (Beckman, J-6M/E, USA) at 1500 x g for 30 min at 4˚C. The supernatant (100µL) was then added to 0.9 mL of assay buffer in new tubes. Then, 100 µL of the diluted samples were made to 300 µL with assay buffer and 100 µL of first antibody was added and incubated overnight at 4˚C. On Day 3, 100 µL of tracer was added, followed by incubation overnight at 4˚C, and on Day 4, 100 µL of second antibody/NRS mixture was added. After incubation overnight at 4˚C, 1 mL of 6% polyethylene glycol (PEG 6000) in assay buffer was added, the samples were centrifuged for 30 min at 1500 x g, and the supernatant aspirated. The activity of the precipitate was then determined on a Gamma Counter (Packard Cobra-II Auto Gamma, Parkard, USA). 3.4.4 Insulin Assay Insulin concentration in plasma was measured in a single assay using the double-antibody RIA method described by Tindal et al. (1978). The minimum detectable concentration was 0.05 µU/mL. The intra-assay CV for low, medium and high quality control samples were 7.3%, 3.2% and 6.1% respectively. The assay included 6 replicates each of three quality control pools. On Day 1, duplicate 100 µL of test plasma samples or standards were diluted to 200 µL with Buffer # 1 + 0.25% BSA. Then 100 µL antiserum was added and the tubes were incubated at 4˚C overnight. On Day 2, 100 µL University of Ghana http://ugspace.ug.edu.gh 32 of the tracer was added, tubes were vortexed and incubation was continued for 48 h at 4˚C. On Day 4, 100 µL of goat anti-guinea pig serum (1:150 in Buffer #1 with 0.25% BSA) and 100 µL of normal guinea pig serum was added and the tubes were vortexed. After incubation overnight at 4˚C, 1.0 mL of 2% polyethylene glycol (PEG 6000) in Buffer # 1was added to the tubes (except total controls) before centrifugation (Beckman, J-6M/E, USA) at 1,500 x g for 25 min at 4˚C. The supernatant was decanted and the pellets were left to dry overnight before the activity of the precipitate was determined on a Gamma Counter (Packard Cobra-II Auto Gamma, Parkard, USA). 3.4.5 Nutritional metabolite analyses The concentrations of the nutritional metabolites in the plasma were measured using the URIT- 810 Semi-auto Biochemical Analyser (Guangzhou Shihai, Medical Equipment Co. Ltd, China) (Plate 3.3). Plasma glucose was measured based on the method of Trinder (1969), while cholesterol was determined based on the enzymatic method of Allain et al. (1974). The total protein concentration, and albumin in the plasma were determined based on the methods of Doumas et al. (1981), and Doumas and Biggs (1972) respectively, while globulin concentration was computed as the difference between the total protein and albumin concentrations (Mapekula et al., 2011). Urea determinations were based on the method by Sampson et al. (1980), while creatinine determinations were according to the method of Ambrose et al. (1983). University of Ghana http://ugspace.ug.edu.gh 33 Plate 3.3: URIT 810 Semi auto Biochemical Analyzer. 3.5 Statistical Analyses The effects of BW, BCS, milk yield, plasma concentrations of the metabolic hormones (GH, IGF-I and insulin), and nutritional metabolites (glucose, cholesterol, total protein, albumin, globulin, urea and creatinine) on resumption of ovarian activity were analysed using the repeated measures analysis of variance procedure of the Statistical Analysis System (SAS, 2003 v.9.1). University of Ghana http://ugspace.ug.edu.gh 34 The model included as fixed effects, treatment (ovarian activity groups), time (weekly or monthly in the case of BW), and their interactions (ovarian activity groups × week). Where the effects were significant, means were separated using the PDIFF procedure of SAS (2003). The Pearson’s partial correlation coefficients were calculated to describe linear relationships among the concentrations of metabolic hormones and plasma metabolites, from week 1 to 9 postpartum, using the SPSS v.16.0 software (SPSS, 2007). University of Ghana http://ugspace.ug.edu.gh 35 CHAPTER FOUR RESULTS 4.1 Resumption of ovarian activity Results on the percentage of cows resuming ovarian activity (Table 4.1) indicate that 37.5% commenced ovarian activity early (by 56 days postpartum), 37.5% commenced ovarian activity later (within 57-112 days postpartum), while 25% failed to commence ovarian cyclicity by 112 days (16 weeks) postpartum. Table 4.1 Resumption of ovarian activity in Friesian-Sanga crossbred cows Ovarian activity groups No. of cows Percentage of cows resuming ovarian activity Early-cycling (≤ 56 days postpartum) 6 37.5 Late-cycling (57-112 days postpartum) 6 37.5 Non-cycling (within 112 days postpartum) 4 25.0 Total 16 100.0 4.2 Daily milk yield, BW and BCS in early-cycling, late-cycling and non-cycling cows Daily milk yield was not significantly different (P>0.05) in the three ovarian activity groups (Table 4.2; Figure 4.1) and there was no treatment x week interaction on daily milk yield (P>0.10). The overall mean daily milk yield was 1.62 ± 0.05 L. The early-cycling, late-cycling and non-cycling cows had similar BW (P>0.05) (Table 4.2). The overall mean BW was 289.9 ± University of Ghana http://ugspace.ug.edu.gh 36 8.33 kg. There was no effect of time (month) or treatment (ovarian activity group) x time (month) interaction on BW. Also, BCS was not significantly different (P>0.05) in early-cycling (5.0 ± 0.27) late-cycling (4.7 ± 0.21) and non-cycling (4.8 ± 0.20) cows (Table 4.2). Overall BCS averaged 4.8 ± 0.23, and there was no effect of time, or treatment x time interaction (P>0.10) on BCS. Table 4.2 Milk yield, body weight, and body condition score in ovarian activity groups in Friesian-Sanga crossbred cattle Parameter Ovarian activity groups postpartum (LSM ± SE) Overall Mean P-value Early cycling ( ≤56 days) n = 6 late cycling (57-112 days) n = 6 non-cycling (within 112 days) n = 4 Milk yield (L/day) 1.53 ± 0.05 1.61 ± 0.05 1.57 ± 0.06 1.62 ± 0.05 0.553 Body weight (kg) Body condition score 301.8 ±7.75 5.0 ± 0.27 286.9 ± 7.75 4.7 ± 0.21 280.0 ± 9.49 4.8 ± 0.20 289.9±8.33 4.8 ± 0.23 0.181 0.096 LSM= Least squares mean SE = Standard error University of Ghana http://ugspace.ug.edu.gh 37 Figure 4.1: Changes in milk yield in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 10 weeks postpartum. All values are least-squares mean ± SEM (n = 16). 4.3 Metabolic hormone concentrations in early-cycling, late-cycling and non-cycling cows The plasma concentrations of IGF-I, insulin and GH in Friesian-Sanga crossbred cows during the postpartum period are presented in Table 4.3. The concentration of IGF-I over weeks 1 to 10 postpartum was higher (P<0.05) in early-cycling (18.7 ± 0.74 ng/mL) than in late-cycling (12.4 ± 0.75 ng/mL), and non-cycling (10.4 ± 0.91 ng/mL) cows (Table 4.3). Generally, IGF-I concentrations increased as the postpartum period progressed after an initial decline in the early- cycling cows, while the late-cycling and non-cycling cows maintained their levels during this University of Ghana http://ugspace.ug.edu.gh 38 period (Figure 4.2), but there was no significant (P>0.10) effect of time or treatment x time interaction. As the postpartum period progressed from week 1 to 10, insulin concentrations remained relatively constant and increased gradually (Figure 4.3), but there was no significant (P>0.10) effect of time or treatment x time interaction. Insulin concentration did not differ (P>0.05) among early-cycling (3.94 ± 0.18 µU/mL), late-cycling (3.98 ± 0.18 µU/mL) and non-cycling (3.49 ± 0.22 µU/mL) cows over weeks 1 to 10 (Table 4.3). Concentrations of GH in early-cycling (1.92 ± 0.21 ng/mL), late-cycling (1.87 ± 0.19 ng/mL), and non-cycling (2.03 ± 0.24 ng/mL) cows were similar (P>0.05) over weeks 1 to 10 (Table 4.3; Figure 4.4). There was no significant (P>0.10) effect of time or treatment x time interaction (P>0.10). Table 4.3 Postpartum plasma metabolic hormone concentrations in early-cycling, late-cycling and non-cycling Friesian-Sanga crossbred cows Parameters Ovarian activity groups (LSM ± SE) Overall Mean P-value Early-cycling (≤56 days) n = 6 Late-cycling (57-112 days) n = 6 Non-cycling (within 112 days) n = 4 IGF-I (ng/mL) 18.7 ± 0.74a 12.4 ± 0.75b 10.4 ± 0.91b 13.8 ± 0.80 0.0001 Insulin (µu/mL) 3.94 ± 0.18 3.98 ± 0.18 3.49 ± 0.22 3.80 ± 0.19 0.1884 GH (ng/ml) 1.92 ± 0.21 1.87 ± 0.19 2.03 ± 0.25 1.94 ± 0.22 0.8741 Means within the same row with different superscripts (a,b) are significantly different (P<0.05) LSM=Least squares mean SE= Standard error University of Ghana http://ugspace.ug.edu.gh 39 Figure 4.2: Changes in plasma IGF-I concentrations in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 10 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 40 Figure 4.3: Changes in plasma insulin concentrations in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 10 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 41 Figure 4.4: Changes in plasma GH concentrations in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 10 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 42 4.4 Nutritional metabolite concentrations in early-cycling, late-cycling and non-cycling cows Glucose, total protein, globulin, urea and creatinine concentrations in the plasma did not differ (P>0.05) in early-cycling, late-cycling or non-cycling cows (Table 4.4; Figure 4.5-4.9). The overall mean glucose, total protein, globulin, urea, and creatinine concentrations in the plasma were 3.26 ± 0.11 mmol/L, 84.7 ± 2.55 mmol/L, 46.6 ± 2.11 mmol/L, 11.4 ± 0.68 mmol/L and 62.3 ± 5.54 mmol/L respectively. Table 4.4 Postpartum plasma nutritional metabolite concentrations in early-cycling, late cycling and non- cycling Friesian-Sanga crossbred cows Parameters Ovarian activity groups(LSM ± SE) Overall Mean P-value Reference Values Early-cycling (≤56 days) Late-cycling (57-112 days) Non-cycling (within 112days) Glucose (mmol/L) 3.38 ± 0.10 3.25 ± 0.10 3.14 ± 0.13 3.26 ± 0.11 0.519 2.2-5.61 Cholesterol (mmol/L) 1.94 ± 0.15b 2.48 ± 0.12a 2.61 ± 0.11a 2.34 ± 0.13 0.003 1.6-5.01 Total Protein (g/L) 86.9 ± 2.38 87.4 ± 2.28 79.8 ± 3.09 84.7 ± 2.55 0.121 67-851,2 Albumin (g/L) 40.7 ± 2.06a 34.4 ± 1.97b 33.6 ± 2.66b 36.2 ± 2.23 0.047 25-381 Globulin (g/L) 44.7 ± 2.85 53.2 ± 2.73 46.2 ± 0.77 46.8 ± 2.11 0.086 30-351 Urea (mmol/L) 11.1 ± 0.63 11.4 ± 0.60 11.7 ± 0.81 11.4 ± 0.68 0.860 3.6-8.91 Creatinine (mmol/L) 60.3 ± 5.12 68.9 ± 4.90 57.8 ± 6.61 62.3 ± 5.54 0.315 44-1941 Means within the same row with different superscripts (a,b) are significantly different (P<0.05) LSM=Least squares mean SE= Standard error Reference values- 1The Merck Veterinary Manual (2010); 2Otto et al. (2000) University of Ghana http://ugspace.ug.edu.gh 43 Figure 4.5: Changes in plasma concentration of glucose in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 44 Figure 4.6: Changes in plasma concentration of Total protein in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 45 Figure 4.7: Changes in plasma concentration of globulin in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 46 Figure 4.8: Changes in plasma concentration of urea in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 47 Figure 4.9: Changes in plasma concentration of creatinine in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). The plasma concentrations of total cholesterol over weeks 1 to 9 were significantly lower (P<0.05) in early- cycling cows than in late-cycling, and non-cycling cows (Table 4.4; Figure 4.10). Time had a significant effect on plasma cholesterol concentrations (P<0.0001); the concentrations were lower in weeks 1(1.84 ± 0.18 mmol/L), and 5 (1.87± 0.17 mmol/L) compared with weeks 3 (2.95 ± 0.16 mmol/L), 7 (2.55 ± 0.16), and 9 (2.50 ± 0.16 mmol/L). However, there was no significant (P>0.10) effect of the treatment x time interaction on total cholesterol concentration. Generally, plasma albumin concentrations declined as lactation University of Ghana http://ugspace.ug.edu.gh 48 progressed from weeks 1 to 9. The plasma concentration of albumin in early-cycling cows was higher (P <0.05) than in late-cycling, and in non-cycling cows (Table 4.4; Figure 4.11). The time of sampling was significant (P=0.0001); concentrations in weeks 1 (46.1 ± 3.13 g/L), 3 (41.9 ± 2.80 g/L), and 5 (35.3 ± 2.97 g/L) were higher than in weeks 7 (31.2 ± 2.80 g/L), and 9 (26.8 ± 2.80 g/L). There was no significant (P>0.10) effect of treatment x time interaction on plasma albumin concentration. Figure 4.10: Changes in plasma concentration of total cholesterol in early-cycling (EC), late- cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). University of Ghana http://ugspace.ug.edu.gh 49 Figure 4.11: Changes in plasma concentration of albumin in early-cycling (EC), late-cycling (LC) and non-cycling (NC) Friesian-Sanga crossbred cows during the first 9 weeks postpartum. All values are least-squares mean ± SEM (n = 16). 4.5 Correlations among metabolic hormones and nutritional metabolites Partial correlation coefficients among concentrations of the metabolic hormones (GH, IGF-I and insulin), and the nutritional metabolites (glucose, cholesterol, total protein, albumin, globulin, urea, and creatinine) (Table 4.5), indicated a positive correlation between IGF-I and insulin (r = 0.328; P<0.01), glucose (r = 0.260; P<0.05), and cholesterol (r = 0.262; P< 0.05). Insulin was positively correlated with glucose (r = 0.502; P<0.10). Glucose was negatively correlated with cholesterol (r = - 0.264; P<0.05), and globulin (r = -0.323; P<0.01), but positively correlated with University of Ghana http://ugspace.ug.edu.gh 50 albumin (r = 0.291; P<0.05). Total protein was positively correlated with globulin (r = 0.706; P<0.01), and urea (r = 0.442; P<0.01). Albumin was negatively correlated with globulin (r = - 0.654; P<0.01), and globulin was positively correlated with urea (r= 0.267; P<0.05). Table 4.5 Partial Correlation coefficients among plasma concentrations of GH, IGF-I, insulin, glucose, cholesterol, total protein, albumin, globulin, urea and creatinine in Friesian- Sanga crossbred cows (n=16) during weeks 1, 3, 5, 7, and 9 postpartum Variable IGF-I Insulin Glucose Cholest Protein Albumin Globulin Urea Creat GH -0.199 0.047 - 0.190 -0.077 -0.129 0.059 -0.052 -0.063 -0.087 IGF-I 0.328** 0.260* 0.262* -0.135 0.070 -0.151 -0.161 -0.020 Insulin 0.502** -0.049 -0.109 -0.028 -0.062 -0.067 -0.149 Glucose - 0.264* -0.156 0.291* -0.323** -0.036 -0.029 Chol 0.082 0.055 0.024 0.161 0.073 Protein 0.074 0.706** 0.442** -0.052 Albumin -0.654** 0.094 0.012 Globulin 0.267* 0.046 Urea 0.001 *P< 0.05 **P<0.01 GH= Growth hormone IGF-I= Insulin-like growth factor-I Chol= Cholesterol; Creat= Creatinine Correlations among metabolic hormones and nutritional metabolites, n = 65 University of Ghana http://ugspace.ug.edu.gh 51 CHAPTER FIVE DISCUSSION Most cows experience a period of negative energy balance due to the inability of insufficient feed intake to meet high energy demands for milk production during early lactation (Butler, 2005; Konigsson et al., 2008; Drackley and Cardoso, 2014). Cows therefore mobilize fat and muscle to support lactation during this period which tends to be associated with changes in concentrations of metabolic hormones and blood metabolites, influencing milk yield and fertility (Wathes et al., 2007). The present study determined the concentrations of some metabolic hormones and nutritional metabolites in the plasma of Friesian-Sanga crossbred cows during the postpartum period. It also evaluated the relationships between these plasma metabolites and the resumption of ovarian activity in these cows. 5.1 Resumption of ovarian activity in Friesian-Sanga crossbred cows The study showed that 37.5% of cows had resumed ovarian activity within 56 days postpartum, 37.5% also resumed ovarian activity between 57 to 112 days postpartum, while 25% of cows failed to resume ovarian activity during the period of study (Table 4.1). The failure of the 25% of the Friesian-Sanga cows to resume ovarian function within 112 days postpartum (16 weeks of lactation), is an indication of extended delay in the resumption of ovarian activity in those cows. This may be due to prolonged period of suckling (6 months) practiced on the station and extensive dependence on natural pasture with no feed supplementation. This limited the ability of lactating cows to meet their nutritional requirements, especially during periods when quality feed was scarce (during the dry season). According to Crowe (2008), poor nutritional status and University of Ghana http://ugspace.ug.edu.gh 52 prolonged suckling tend to delay postpartum resumption of ovulation in cattle by reducing LH secretion and pulsatility. Obese et al. (2009) reported that prolonged postpartum anoestrous periods in cows is a major infertility problem resulting in extended calving to conception and calving intervals in indigenous cattle breeds in Ghana. The authors reported prolonged postpartum anoestrous periods ranging from 101 to 107 days, resulting in extended calving intervals from 431 to 444 days, in Sanga cows raised in smallholder farms on the Accra Plains. 5.2 Daily milk yield, BW and BCS and resumption of ovarian activity The mean daily milk yield of 1.62 L recorded in this study was similar to the 1.42 L recorded for Friesian-Sanga crossbred cows in an earlier study on the same station (Darfour-Oduro et al., 2010), but higher than the 1.06 L recorded for the Sanga by the same authors. The higher milk production of the Friesian-Sanga crossbred compared with the local Sanga may be due to the advantage afforded by heterosis through crossbreeding, as indicated in a review by Galukande et al. (2013). The BCS of an animal reflects its nutritional and health status, and is useful for predicting its reproductive performance (Pryce et al., 2001). BCS at key periods in lactation, as well as BCS changes have been associated with the resumption of oestrus cycles and reproductive success in cows (Pryce et al., 2001). Early-cycling cows in the present study had numerically better body condition score (5.0) than the late (4.7), and non-cycling (4.8) cows. Low BCS after calving has been reported to increase the risk of delayed ovulation in cows (Montiel and Ahuja, 2005; Tamadon et al., 2011). The overall mean BCS of 4.8 ± 0.23 obtained for cows in this study suggest cows were in poor to medium (moderate) body condition on the 9-point score of Nicholson and Butterworth (1986). University of Ghana http://ugspace.ug.edu.gh 53 5.3 Metabolic hormone concentrations and resumption of ovarian activity The plasma concentrations of IGF-I in postpartum cattle have been correlated with reproductive performance (Patton et al., 2007; Falkenberg et al., 2008). In the present study, IGF-I concentrations were higher in early-cycling than in late, and non-cycling cows (Table 4.3). Elevated circulating concentrations of IGF-I during the postpartum period have been associated with the earlier resumption of ovarian activity of cows in pasture-based systems (Obese et al., 2012; Samadi et al., 2013). High levels of circulating IGF-I is reported to enhance follicular cell responsiveness to LH which in turn increases follicular oestradiol production and thus hasten ovulation (Diskin et al., 2003; Crowe, 2008; Peter et al., 2009). The plasma concentrations of IGF-I obtained during the postpartum period in the present study were lower than the range of values 14.7 to 23.2 ng/mL obtained from week 1 to week 10 in suckled Sanga cows grazing natural pasture in Ghana (Obese et al., 2012). It was also lower than the range of values 40.9 ± 0.5 to 53.3 ± 10.1 ng/mL obtained between calving and day 40 postpartum for suckled Angus x Nelore cows grazing natural pasture in Brazil (Schneider et al., 2010). Breed differences and nutritional status of cows may account for the above differences. The Friesian-Sanga crossbred may have had difficulty in meeting their higher nutritional requirements in depending solely on natural pasture without any feed supplementation. Systemic concentrations of IGF-I in cows are known to be directly influenced by nutrient intake (Ciccioli et al., 2003). Insulin serves as a metabolic signal influencing LH release by the anterior pituitary (Monget and Martin, 1997), and also regulates ovarian responsiveness to gonadotrophins (Diskin et al., 2003). University of Ghana http://ugspace.ug.edu.gh 54 Lower levels of insulin may thus suppress LH release and consequently, delay ovulation. Sinclair et al. (2002) observed that postpartum anoestrous beef cows with low plasma concentrations of insulin were unable to ovulate a dominant follicle compared with cows with higher plasma concentrations of insulin. In cattle, the main effects of GH on reproduction appear to be operated through its regulatory effects on hepatic IGF-I synthesis and secretion (Gong, 2002; Lucy et al., 1999). In the present study, however, insulin and GH concentrations did not influence the early resumption of ovarian activity as the concentrations were similar in early-cycling, late-cycling and non-cycling cows. The plasma insulin concentrations obtained during the postpartum period was similar to the range of 3 to 4 µU/mL reported by Obese et al (2012) for Sanga cows grazing natural pasture on the Accra Plains, but lower than the range of 4.5 to 8 µU/mL reported for Zebu crossbred cows (Samadi et al., 2013). Cows in the study by Samadi et al. (2013) grazed on improved pastures and this may account for the higher concentrations of insulin in that study. Insulin concentration is known to reflect energy status and dietary adequacy (Drackley and Cardoso, 2014). 5.4 Nutritional metabolite concentrations and resumption of ovarian activity The determination of nutritional metabolite concentrations provides information on the adequacy of nutrient supply in relation to nutrient utilization (Chester-Jones et al., 1990). They indicate the extent of metabolism of energy, proteins and other nutrients in animals, giving instant indication of the animal’s nutritional status at any particular point in time (Pambu-Gollah et al., 2000). The plasma glucose, total protein, globulin, urea, and creatinine concentrations in the present study did not differ among the early-cycling, late-cycling or non-cycling cows, indicating no relationship with the resumption of ovarian activity in the Friesian-Sanga crossbred cows University of Ghana http://ugspace.ug.edu.gh 55 studied. The resumption of ovarian activity was, however, associated with concentrations of total cholesterol and albumin (Table 4.4). Total cholesterol concentrations in the plasma were higher in late and non-cycling cows than in early-cycling cows. Perhaps, increased lipolysis in these two groups of cows due to their lower glucose concentrations resulted in increased plasma concentrations of low density lipoproteins which is associated with increased rate of cholesterol synthesis, as reported in some studies (Ahmad et al., 2004; Saleh et al, 2011). The overall mean plasma cholesterol level of 2.34 mmol/L recorded was similar to the value of 2.47mmol/L obtained by Damptey et al. (2014) for Sanga cows grazing extensively on natural pasture on the Accra plains. Elevated plasma albumin concentrations in this study were associated with early resumption of ovarian activity. This may be due to the better BCS in the early-cycling (5.0) compared with late- cycling (4.7) or non-cycling (4.8) cows. Blood albumin concentration is reported to reflect good nutritional condition and hence good BCS in cattle (Kaneko et al., 1997; Coppo, 2004). The concentrations of the nutritional metabolites measured in the plasma in this work were within normal ranges reported for cows (Otto et al., 2000; The Merck Veterinary Manual, 2010) apart from the higher values recorded for globulin (46.81) and urea (11.4) (Table 4.4). This suggests that cows in the production system may have been prone to infections and also experienced energy deficiencies. Circulating concentrations of globulin gives indication of an animal’s immune state, and its responses to fighting diseases and infections. Thus, circulating globulin concentrations are usually high during parasitic and microbial infections (Kapele et al., 2008). Urea concentrations in the blood are also elevated when there is energy deficiency in University of Ghana http://ugspace.ug.edu.gh 56 cows, limiting microbial protein synthesis (Murphy, 1999). It has been reported (Okantah et al., 1999; Obese et al., 2009) that cattle grazing extensively on natural pasture in the smallholder production system in Ghana experience inadequate nutrition and fluctuating nutrient supply especially in the dry season, leading to reduced growth rates and milk production and delayed resumption of ovarian cycles. According to Osafo et al. (2013), owing to the inherent nutrient deficiencies in native grasses, it cannot sustain effective animal production when fed alone. Therefore, the introduction of appropriate supplementary feedstuffs would be an important step to enhance the productivity of cattle under smallholder and pastoral production systems in Ghana. 5.5 Relationships among metabolic hormones and nutritional-related metabolites In the present study, plasma concentrations of IGF-I, insulin, glucose and cholesterol were positively and significantly correlated during week 1 to 9 postpartum, suggesting these metabolites are likely mediators of energy induced alterations in ovarian function during the postpartum period in cows. Insulin enhances the synthesis of IGF-1 in the liver in response to elevated concentrations of GH to increase oestradiol production by the dominant follicle, resulting in increased LH receptors for ovulation and corpus luteum development (Lucy, 2000; Garnsworthy et al., 2008). Thus, higher circulating IGF-I, insulin and glucose concentrations stimulate GnRH and LH release, hastening ovulation postpartum (Diskin et al., 2003; Wetterman et al., 2003; Crowe et al., 2014). Concentrations of IGF-I have been reported to be directly related to concentrations of insulin and glucose in beef (Ciccioli et al., 2003; Damptey et al., 2013) and dairy (Zulu et al., 2002) cows. There was a significant positive correlation between insulin and glucose in the present study. Insulin is primarily involved in stimulating glucose University of Ghana http://ugspace.ug.edu.gh 57 uptake by cells (Wetteman et al., 2003). In early lactation when cows are in negative energy balance, they may develop ketosis and experience depressed insulin and glucose level with elevated ketones and free fatty acids as well as cholesterol in the blood (Schwalm and Schultz, 1976). Damptey (2013) reported a significant correlation between insulin and glucose in Sanga cows during the postpartum period. Glucose was significantly and positively correlated with albumin, but negatively correlated with cholesterol and globulin. Glucose is utilized by all animal cells to produce energy (Butler, 2014). The positive correlation between glucose and albumin suggest nutrient intake will regulate glucose and albumin in the same direction whilst regulating glucose, cholesterol and urea in the opposite direction. Damptey et al. (2013) also reported positive correlation between glucose and albumin concentration, but negative correlation between glucose and urea in Sanga cows grazing extensively on natural pasture in the Accra Plains. The positive significant correlation among plasma total protein, globulin and urea indicates that changes in plasma total protein levels invariably affect plasma globulin and urea concentrations during ovarian activity in the postpartum period. The significant negative correlation between plasma albumin and globulin concentrations may indicate the opposite influence of ovarian activity on these metabolites during this period. Plasma globulin and urea concentration was positive and significantly correlated, indicating these metabolites are likely affected in the same direction during ovarian activity in the postpartum period. University of Ghana http://ugspace.ug.edu.gh 58 CHAPTER SIX CONCLUSIONS AND RECOMMENDATIONS 6.1 CONCLUSIONS 1. Concentrations of the metabolic hormones GH, IGF-I and insulin were low, and body condition score was poor to medium (moderate), indicating poor metabolic and nutritional status in the Friesian-Sanga crossbred cows. This suggests that the prolonged postpartum anoestrous intervals in the Friesian-Sanga crossbred cows is associated with reduced concentration of these metabolic hormones which play important roles in the commencement of ovulation, postpartum. 2. Early resumption of ovarian activity postpartum in Friesian-Sanga cows was associated with higher plasma concentrations of IGF-I and albumin, but lower cholesterol concentrations. 3. The concentrations of most of the nutritional metabolites measured were within acceptable physiological ranges reported for cows. 4. Positive or negative relationships were observed among the concentrations of some of the metabolites measured. The positive relationships between changes in some energy (eg glucose) and protein-related metabolites (eg, albumin), and metabolic hormones associated with the resumption of ovarian activity (IGF-I and insulin) supports the view that the effect of alterations in energy balance and/or protein balance on postpartum ovarian function could be mediated through changes in the secretory patterns of these metabolic hormones. University of Ghana http://ugspace.ug.edu.gh 59 6.2 RECOMMENDATIONS 1. The nutritional and metabolic status of the cows could be improved through the introduction of supplementary feed packages under this production system. This will enhance energy and protein availability for efficient microbial protein synthesis, effective feed utilization and consequently, improve growth and body condition. Also the production and secretion of IGF-I and insulin which play major roles in the resumption of ovarian activity postpartum will be improved. 2. Early weaning should be introduced in the production system in order to reduce the effect of the suckling stimulus on extension of postpartum anoestrous periods, thereby reducing calving intervals. 3. Studies to evaluate the effects of feed supplementation and early weaning on metabolic hormones and related metabolites which affect ovarian function in cows should be undertaken. University of Ghana http://ugspace.ug.edu.gh 60 REFERENCES Abeygunawardena, H. and Dematawewa, C. M. B. 2004. Pre-pubertal and postpartum anestrus in tropical Zebu cattle. Animal Reproduction Science 82-83: 373-387. Abou-Tarboush, H.M. and Dawood, A. A. 1993. Cholesterol and fat contents of animal adipose tissues. Food Chemistry 46(1): 89-93. Agenas, S, Heath, M.F., Nixion, R.M., Wilkinson, J.M. and Phillips, C.J. 2006. Indicators of under nutrition in cattle. Animal Welfare 15(2): 149-160. Ahmad, I., Lodhi, L.A., Qureshi, Z.I. and Younis M. 2004. Studies on blood glucose, total protein, urea and cholesterol levels in cyclic, non-cyclic and endometritic crossbred cows. Pakistanian Veterinary Journal 24: 92-94. Allain, C.C., Poon, L.S., Chan, C.S.G., Richmond, W. and Fu, P.C. 1974. Enzymatic determination of total serum cholesterol. Clinical Chemistry 20: 470–475. Ambrose, R.T., Ketchum, D.F. and Smith, J.W. 1983. Creatinine determined by “High Performance” Liquid Chromatography. Clinical Chemistry 29, 256-259. Baumrucker, C.R. and Erondu, N.E. 2000. Insulin-like growth factor (IGF) system in the bovine mammary gland and milk. Journal of Mammary Gland Biology and Neoplasia 5(1): 53- 64. Beam, S.W. and Butler,W.R. 1997. Energy balance and ovarian follicle development prior to first ovulation postpartum in dairy cows receiving three levels of dietary fat. Biology of Reproduction 56: 133–142. Beam, S.W. and Butler, W.R. 1999. Effects of energy balance on follicular development and first ovulation in postpartum dairy cows. Journal of Reproduction and Fertility (Supplement) 54: 411-424. University of Ghana http://ugspace.ug.edu.gh 61 Bearden, H. J. and Fuquay, J. W. 1984. Applied Animal Reproduction. 2nd Edition. Reston Publication Co. Inc., Reston, Virginia. Pp 63-296. Bell, A.W. and Buamans, D. E. 1997. Adaptations of glucose metabolism during pregnancy and lactation. Journal of Mammary Gland Biology and Neoplasia 2: 265-277. Bellows, R. A. and Short, R. E. 1978. Effects of precalving feed level on birth weight, calving difficulty and subsequent fertility. Journal of Animal Science 46:1522-1528. Bellows, R. A., Short, R. E., Staigmiller, R. B. and Milmine, W. L. 1988. Effects of induced parturition and early obstetrical assistance in beef cattle. Journal of Animal Science 66: 1073-1080. Berry, D. P., Buckley, F., Dillon, P., Evans, R.D., Rath, M. and Veerkamp, R.F. 2003. Genetic relationships among body condition score, body weight, milk yield, and fertility in dairy cows. Journal of Dairy Science 86: 2193–2204. Berry, D.P., Roche, J.R. and Coffey, M.P. 2007. Body Condition Score and Fertility– More Than Just a Feeling. Fertility in Dairy Cows – Bridging the gaps. Liverpool Hope University, Liverpool, UK, pp 107–118. Blache, D., Celi, P., Blackberry, M.A., Dynes, R.A. and Martin, G.B. 2003. Decrease in voluntary feed intake and pulsatile luteinizing hormone secretion after intracerebroventricular infusion of recombinant bovine leptin in mature male sheep. Journal of Reproduction, Fertility and Development 12: 373–381. Block, S.S., Butler, W.R., Ehrhardt, R.A., Bell, A.W., Van Amburgh, M.E. and Boisclair, Y.R. 2001. Decreased concentration of plasma leptin in periparturient dairy cows is caused by negative energy balance. Journal of Endocrinology 171:339-348. University of Ghana http://ugspace.ug.edu.gh 62 Bossis, I., Welty, S.D., Wettemann, R.P., Vizcarra, J.A., Spicer, L.J. and Diskin, M.G. 1999. Nutritionally induced anovulation in beef heifers: ovarian and endocrine function preceding cessation of ovulation. Journal of Animal Science 77: 1536–1546. Breier, B.H., Gallaher, B.W. and Gluckman, P.D. 1991. Radioimmunoassay for insulin-like growth factor-1: solution to some potential problems and pitfalls. Journal of Endocrinology 128: 345–357. Brinks, J. S., Olson, J. E. and Carroll, E. J. 1973. Calving difficulty and its association with subsequent productivity in Herefords. Journal of Animal Science. 36: 11-17. Britt J.H., Armstrong J.D., Moore K.L. and Sesti L.A.C. 1993. Involvement of opioids in regulation of LH secretion during lactational and nutritional-induced anestrus in pigs and cattle. In: Parvizi N (ed.) Opioids in Farm Animals Land-Wirtschaftsverlag Munster. pp 35-54. Buckley, F., O’Sullivan, K., Mee, J.F., Evans, R.D. and Dillon, P. 2003. Relationships among milk yield, body condition, cow weight, and reproduction in spring-calved Holstein- Friesians. Journal of Dairy Science 86: 2308–2319. Butler, S.T. 2014. Nutritional management to optimize fertility of dairy cows in pasture-based systems. Animal 8 (Supplement 1): 15-26. Butler, W.R. 2000. Nutritional interactions with reproductive performance in dairy cattle. Animal Reproduction Science 60-61: 449-457. Butler, W.R. 2003. Energy balance relationships with follicular development, ovulation and fertility in postpartum dairy cows. Livestock Production Science 83: 211–218. Butler, W.R. 2005. Inhibition of ovulation in the postpartum cow and the lactating sow. Livestock Production Science 98: 5–12. University of Ghana http://ugspace.ug.edu.gh 63 Cameron, B., Rahal, J.O. and Mayo, K.E., 1998. Cellular localization and hormonal regulation of follicle-stimulating hormone and luteinizing hormone receptor messenger RNAs in the rat ovary. Molecular Endocrinology 5: 1405–1417. Chagas, L.M., Bass, J.J., Blache, D., Burke, C.R., Kay, J.K., Lindsay, D.R., Lucy, M.C., Martin, G.B., Meier, S., Rhodes, F.M., Roche, J.R., Thatcher, W.W. and Webb, R. 2007. Invited review: new perspectives on the roles of nutrition and metabolic priorities in the subfertility of high-producing dairy cows. Journal of Dairy Science 90: 4022–4032. Chester-Jones, H., Fontenot, J.P. and Veit, H.P. 1990. Physiological and pathological effects of feeding high levels of magnesium to steers. Journal of Animal Science 68: 4400-4413. Ciccioli, N.H., Wettemann, R.P., Spicer, L.J., Lents, C.A., White, F.J. and Keisler, D.H. 2003. Influence of body condition at calving and postpartum nutrition on endocrine function and reproductive performance of primiparous beef cows. Journal of Animal Science 81: 3107–3120. Clarke, I.J. and Henry, B.A. 1999. Leptin and Reproduction. Review of Reproduction 4: 48-55. Cohick, W.S. 1998. Role of the insulin-like growth factors and their binding proteins in lactation. Journal of Dairy Science 81: 1769-1777. Coles, E.H. 1996. Veterinary Clinical Pathology, 4th Edition, W.B. Saunders Company, London. Coppo, J.A. 2004. Biochemistry demonstration of malnutrition state in early weaned half-bred Zebu calves. Review of Investigative Agropecuarias 33: 81-100. Crowe, M.A. 2008. Resumption of ovarian cyclicity in post-partum beef and dairy cows. Reproduction in Domestic Animal 43 (Suppl. 5): 20–28. Crowe, M.A., Diskin, M.G. and Williams, E.J. 2014. Parturition to resumption of ovarian cyclicity: comparative aspects of beef and dairy cows. Animal 8 (Supplement 1): 40-53. University of Ghana http://ugspace.ug.edu.gh 64 Cummins, S.B., Waters, S.M., Evans, A.C., Lonergan, P. and Butler, S.T. 2012. Genetic merit for fertility traits in Holstein cows: III. Hepatic expression of somatotropic axis genes during pregnancy and lactation. Journal of Dairy Science 95: 3711-3721. Dahl, G.E., Buchanan, B.A. and Tucker, H.A. 2000. Photoperiodic effects on dairy cattle: A review. Journal of Diary Science 81: 885-893. Dahl, G.E., Elsasser, T.H., Capuco, A.V., Erdman, R.A. and Peters, R.R. 1997. Effects of long daily photoperiod on milk yield and circulating insulin-like growth factor-1 (IGF-1). Journal of Dairy Science 80: 2784-2789. Damptey, J.K. 2012. Relationships among some plasma metabolites and resumption of ovarian function in Sanga cows. M.Phil Thesis Department of Animal Science, University of Ghana, Legon. Damptey, J.K., Obese, F.Y., Aboagye, G.S., Ayim- Akonor, M and Ayizanga, R.A. 2014. Blood metabolite concentrations and postpartum resumption of ovarian cyclicity in Sanga cows. South African Journal of Animal Science 44:10-17. Damptey, J.K., Obese, F.Y., Aboagye, G.S. and Ayizanga, R.A. 2013. Correlations among concentrations of some metabolic hormones and nutritionally-related metabolites in beef cows. Online Journal of Animal and Feed Research 3: 176–180. Darfour-Oduro, K.A., Sottie, E.T., Hagan B.A. and Okantah S.A. 2010. Milk yield and lactation length of Ghana Sanga and its crosses with the Friesian raised under agropastoral system. Tropical Animal Health Production 42: 349–356. Diskin, M.G., Mackey, D.R., Roche, J.F. and Sreenan, J.M. 2003. Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle. Animal Reproduction Science 78: 345-370. University of Ghana http://ugspace.ug.edu.gh 65 Doornbos, D. E., R. A. Bellows, P. J. Burfening and B. W. Knapp. 1984. Effects of dam age, prepartum nutrition and duration of labor on productivity and postpartum reproduction in beef females. Journal of Animal Science 59:1-10 Doornenbal, H., Tong, A.K.W. and Murray, N.L. 1988. Reference values of blood parameters in beef cattle of different ages and stages of lactation. Canadian Journal of Veterinary Research 52(1): 99-105. Doumas, B., Bayse, D.D., Carter, R.J., Peters, T. and Schafer, R. 1981. A candidate reference method for determination of total protein in serum: 1. Development and validation. Clinical Chemistry 10: 42-50. Doumas, B.T. and Biggs, H.G. 1972. Determination of serum albumin. Standard Methods in Clinical Chemistry 7: 175–188. Drackley, J.K. and Cardoso, F.C. 2014. Prepartum and postpartum nutritional management to optimize fertility in high-yielding dairy cows in confined TMR systems. Animal 8 (supplement 1): 5–14. Dunn. T. G., J. E. Ingalls, D. R. Zimmerman and J. N. Wiltbank. 1969. Reproductive performance of 2-year-old Hereford and Angus heifers as influenced by pre- and post- calving energy intake. Journal of Animal Science 29:719. Dyer, C. J., Simmons, J.M., Matteri, R.L. and Keisle, D.H. 1997. Leptin receptor mRNA is expressed in ewe anterior pituitary and adipose tissues and is differentially expressed in hypothalamic region of well-fed and feed-restricted ewes. Domestic Animal Endocrinology 14: 119-128. University of Ghana http://ugspace.ug.edu.gh 66 Edfers-Lilja, C.C., van Amburgh, M. and Butler, W.R. 1980. Alterations of pH in response to increased dietary protein in cattle are unique to the uterus. Journal of Animal Science 71: 702-706. Ehrhardt, R.A., Slepetis, R.M., Siegal-Willott, J., Van Amburgh, M.E., Bell, A.W. and Boisclair, Y.R. 2000. Development of a specific radioimmunoassay to measure physiological changes of circulating leptin in cattle and sheep. Journal Endocrinology 166: 519–528. Esposito, G., Irons, P.C and Webb, E.C. 2014. Interactions between negative energy balance, metabolic diseases, uterine health and immune response in transition dairy cows. Animal Reproduction Science 144: 60-71. Falkenberg, U., Haertel, J., Rotter, K., Iwersen, M., Arndt, G. and Heuwieser, W. 2008. Relationships between the concentration of IGF-I in serum in dairy cows in early lactation and reproductive performance and milk yield. Journal of Dairy Science 91: 3862-3868. Fonseca, F.A., Britt, J.H., McDaniel, B.T., Wilk, J.C. and Rakes, A.H. 1983. Reproductive traits of Holstein and Jerseys: effects of age, milk yield, and clinical abnormalities on involution of cervix and uterus, ovulation, estrous cycles, detection of estrus, conception rate and days open. Journal of Dairy Science 66: 1128-1147. Frandson, R.D., Wilke R.D and Fails, A.D. 2009. Anatomy and Physiology of Farm Animals 7th Edition, Wiley-Blackwell, U.S.A. Galukande, E., Mulindwa, H., Wurzinger, M., Roschinsky, R., Mwai, A.O. and Solkner, J. 2013. Animal Genetic Resource 52: 111-125. University of Ghana http://ugspace.ug.edu.gh 67 Garel. J. P., D. Gauthier, M. and 'Ibimonier, J. 1987. Influence of photoperiod on the post-partum changes in live weight and ovarian function in suckled cow. Reproduction, Nutrition and Development 27: 305-312. Garnsworthy, P.C., Sinclair, K.D. and Webb, R. 2008. Integration of physiological mechanisms that influence fertility in dairy cows: Animal 2: 1144-1152. Giuliodori, M.J., Delavaud, C., Chilliard, Y., Becu-Villalobos, D., Lacau-Mengido, I. and dela Sota, R.L. 2011. High NEFA concentrations around parturition are associated with delayed ovulations in grazing dairy cows. Livestock Science 141: 123-128. Gluckman, P.D., Johnson-Barrett, J.J., Butler, J.H., Edgar, B.W., Gunn, T.R. 1983. Studies in insulin- like growth factor-I and-II by specific radioligand assays in umbilical cord blood. Clinical Endocrinology 19: 405–413. Gong, J.G., Lee, W.J., Garnsworthy, P.C. and Webb, R. 2002. Effect of dietary induced increases in circulating insulin concentrations during the early postpartum period on reproductive function in dairy cows. Reproduction 123: 419–427. Greenwood, P., Hunt, A., Slepetis, R., Finnerty, K., Alston, C., Beermann, D. and Bell, A.W. 2002. Effects of birth weight and postnatal nutrition on neonatal sheep. III. Regulation of energy metabolism. Journal of Animal Science 80: 2850-2861. Hafez, E. S. E. and Hafez, B. 2000. Reproduction in Farm Animals. 7th Edition, 2000. Lippincott Williams and Wilkins, Baltimore, USA. Hansen. P. J., D. H. Baik, J. J. Rutledge and E. R. Hauser. 1982. Genotype x environmental interactions on reproductive traits of bovine females. II. Postpartum reproduction as influenced by genotype, dietary regimen, level of milk production and parity. Journal of Animal Science 55:1458-1472. University of Ghana http://ugspace.ug.edu.gh 68 Hess, B.W., Lake, S.L., Scholljegerdes, E.J., Weston, T.R., Nayigihugu, V., Molle, J.D.C. and Moss, G.E. 2005. Nutritional controls of beef cow reproduction. Journal of Animal Science 83: E90-E106. Imakawa, K., Day, M.L., Garcia-Winder, M., Zalesky, D.D., Kittok, R.J., Schanbacher, K.D. and Kinder, J.E. 1986. Endocrine changes during restoration of oestrous cycles following induction of anoestrous by restricted nutrient intake in beef heifers Journal of Animal Science 63: 565–571. Jolly, P.D., McDougall, S., Fitzpatrick, L.A., Macmillan, K.L., and Entwistle, K.W. 1995. Physiological effects of under-nutrition on postpartum anoestrous in cows. Journal of Reproduction and Fertility (Supplement) 49: 477-492. Jones, J.I. and Clemmons, D.R. 1995. Insulin-like growth factors and their binding proteins: biological actions. Endocrine Reviews 16:3-33. Jorritsma, R., Wensing, T., Kruip, T. A., Vos, P. L. and Noordhuizen, J. P. 2003. Metabolic changes in early lactation and impaired reproductive performance in dairy cows. Veterinary Research 34: 11-26. Kadokawa, H., Blache, D., Yamada, Y., Martin, G.B. 2000. Relationships between changes in plasma concentrations of leptin before and after parturition and the timing of first post- partum ovulation in high producing Holstein dairy cows. Reproduction in Fertility Development 12: 405–411. Kaneko, J.J., Harvey, J.W. and Bruss, M.L. 1997. Clinical Biochemistry of Domestic Animals (5th Edition). Academic Press, Inc., New York, USA. Kapele, P.M., Japtap, D.G., Badukale, D.M. and Sahatpure, S.K. 2008. Serum total proteins and serum total cholesterol levels in Gaolao cattle. Veterinary World 1, 115–116. University of Ghana http://ugspace.ug.edu.gh 69 Karikari, P.K., Gyawu, P., Asare, K. and Yambillah, S.S. 1995. The reproductive response of N’dama cows to brewers’ spent grain supplementation in a hot humid environment. Tropical Agriculture 72: 315-318. Kawashima, C., Sakaguchi, M., Suzuke, T., Sasamoto, Y., Takahashi, Y., Matsui, M. and Miyamoto, A. 2007. Metabolic profiles in ovulatory and anovulatory primiparous dairy cows during the first follicular wave postpartum. Journal of Reproduction Development 53: 113-120. King, G. J. and Macleod, G. A. 1983. Reproductive function in beef cows calving in the spring or fall. Animal Reproduction Science 6:255-266. Konigsson, K., Savoini, G., Govoni, N., Invernizzi, G., Prandi, A., Kindahl, H. and Veronesi, M. C. 2008. Energy balance, leptin, NEFA and IGF-I plasma concentrations and resumption of postpartum ovarian activity in Swedish red and white breed cows. Acta Veterinaria Scandinavica 50: 1–7. Lavon, Y., Ezra, E., Leitner, G. and Wolfenson, D. 2011. Association of conception rate with pattern and level of somatic cell count elevation relative to time of insemination in dairy cows. Journal of Dairy Science 94: 4538-4545. LeBlanc, S. J., Duffield, T.F., Leslie, K.E., Bateman, K.G., Keefe, G.P., Walton, J.S. and Johnson, W.H. 2002. Defining and diagnosing postpartum clinical endometritis and its impact on reproductive performance in dairy cows. Journal of Dairy Science 85: 2223- 2236. Le Roith, D., Bondy, C., Yakar, S., Liu, J.L. and Butler, A. 2001. The Somatomedin Hypothesis. Endocrine Reviews 22: 53-74. University of Ghana http://ugspace.ug.edu.gh 70 Leng, R.A. 1992. Improving ruminant production and reducing methane emissions by strategic supplementation. Report to the U.S. Environmental Protection Agency. Washington, D.C. EPA. Lindsay, D.B., Hunter, R.A., Gazzola, C., Spiers, W.G. and Sillence, M.N. 1993. Energy and growth. Australian Journal of Agricultural Research 44:875 – 889. Lucy, M.C. 2000. Regulation of ovarian follicular growth by somatotropin and insulin-like growth factors in cattle. Journal of Animal Science 83: 1635- 1647. Lucy, M.C. 2001. Reproductive loss in high-producing dairy cattle: where will it end? Journal of Dairy Science 84: 1277–1293. Lucy, M.C., Bilby, C.R., Kirby, C.J., Yuan, W. and Boyd, C.K. 1999. Role of growth hormone in development and maintenance of follicles and corpora lutea. Journal of Reproduction Fertility (Supplement.) 54: 49–59. Macial, S.M., Chamberlian, C.S., Wettemann, R.P. and Spicer, L.J. 2001. Dexamethasone influences endocrine and ovarian function in dairy cattle. Journal of Dairy Science 84: 1998-2009. Mackey, D.R.,Wylie, A.R.G., Sreenan, J.M., Roche, J.F. and Diskin, M.G. 2000. The effect of acute nutritional change on follicle wave turnover, gonadotropin, steroid concentration in beef heifers. Journal of Animal Science 78: 429–442. Macmillan, K. L. 1983. A review of developments in nutrition as it relates to fertility in cattle. New Zealand Veterinary Journal 18: 61-65. Mapekula, M., Mapiye, C. and Chimonyo, M. 2011. Changes in metabolites concentration in Nguni and crossbred calves on natural pasture. Asian-Australasian Journal of Animal Science 24: 1569–1576. University of Ghana http://ugspace.ug.edu.gh 71 McArt, J.A., Nydam, D.V. and Oetzel, G.R. 2012. Epidemiology of subclinical ketosis in early lactation dairy cattle. Journal of Dairy Science 95: 5056-5066. McDougall, S. 1994. Postpartum anoestrous in pasture grazed New Zealand dairy cows. PhD Thesis. Massey University. Palmerston North, New Zealand. McDougall, S., Blanche, D. and Rhodes, F. M. 1995b. Factors affecting conception and expression of oestrus in anoestrous cows treated with progesterone and oestradiol benzoate. Animal Reproduction Science 88: 203-214. McDougall, S., Burke, C.R., Macmillan, K.L. and Williamson, N.B. 1995a. Patterns of follicular development during periods of anovulation in pasture-fed dairy cows after calving. Research in Veterinary Science 58: 212-216. McDougall, S., Macmillan, K.L. and Williamson, N. B. 1998. Factors associated with a prolonged period of postpartum anoestrous in pasture-fed dairy cattle. Proceedings of World Association of Buiatrics 20: 657-662. Mellado, M. and Reyes, C. 1994. Associations between peri-parturient disorders and reproductive efficiency in Holstein cows in Northern Mexico. Preventive Veterinary Medicine 19: 203-212. Mihm, M. 1999. Delayed resumption of cyclicity in postpartum dairy and beef cows. Reproduction in Domestic Animals 34: 277-284. Miller, S.C., Bruce, E.L., Heather, L.T., Perry, J. B. and Latimer, K.S. 2004. A brief review of creatinine concentration. Veterinary Clinical Pathology Clerkship Program, College of Veterinary Medicine, University of Georgia, Athens, Georgia 3060-7388. University of Ghana http://ugspace.ug.edu.gh 72 Monget, P. and Martin, G.B. 1997. Involvement of insulin-like growth factors in the interactions between nutrition and reproduction in female mammals. Human Reproduction 12 (Supplement 1): 33–52. Montiel, F. and Ahuja, C. 2005. Body condition and suckling as factors influencing the duration of postpartum anestrus in cattle: a review. Animal Reproduction Science 85: 1-26. Morrow, D.A. 1986. Current therapy in Theriogenology. 2nd Edition. W.B. Saunders Company, Philadelphia, USA. Pp. 1104. Mukasa-Mugerwa, E. 1989. A Review of Reproductive Performance of Female Bos indicus (Zebu) cattle. ILCA Monograph No. 6, Addis Ababa, Ethiopia. Pp 1-134. Mukasa-Mugerwa, E., Tegegne, A. and Franceschini, R. 1991. Influence of suckling and continuous cow-calf association on the resumption of postpartum ovarian function in Bos indicus cows monitored by plasma progesterone profiles. Reproduction Nutrition and Development 71:241-247. Murphy J.J. 1999. The effects of increasing the proportion of molasses in the diet of milking cows on milk production and composition. Animal Feed Science and Technology 79: 189-198. Mwaaga, E. S. and Janowski, T. 2000. Anoestrous in Dairy cows: Causes, prevalence and clinical forms. Reproduction in Domestic Animals 35: 193-200. Ndlovu, T., Chimonyo, M., Okoh, A.I., Muchenje, V., Dzama, K. and Raats, J.G. 2007. Review: Assessing the nutritional status of beef cattle: current practices and future prospects. African Journal of Biotechnology 6(24): 2727-2734. Nicholson M.J., Butterworth M.H. 1986. A guide to condition scoring of Zebu cattle. International Livestock Centre for Africa, Addis Ababa. 29 pp. University of Ghana http://ugspace.ug.edu.gh 73 Obese, F. Y., Okantah, S. A., Oddoye, E. O. K. and Gyawu, P. 1999. Post-partum reproductive performance of Sanga cattle in small-holder peri-urban dairy herds in the Accra plains of Ghana. Tropical Animal Health and Production 31: 181 – 190. Obese, F.Y., Acheampong D.A. and Darfour-Oduro, K.A. 2013. Growth and reproductive traits of Friesian x Sanga crossbred cattle in the Accra Plains of Ghana. African Journal of Food, Agriculture Nutrition Development 13: 7357-7371. Obese, F.Y., Damptey J.K., Aboagye G.S., Ayizanga, R.A. and Owusu–Ntumy, D. 2012. Relationships between body condition score, milk yield, insulin-like growth factor-I concentration and resumption of ovarian activity in beef cows. Bulletin of Animal Health and Production in Africa 60: 445-452. Obese, F.Y., Darfour-Oduro, K.A. and Adu, E.K. 2009. Effects of extended postpartum anoestrous period on reproductive performance of indigenous beef cattle raised on small- holder farms in Ghana: An overview. Ghanaian Journal of Animal Science 4 :( 1) 1-11. O’Callanghan, D. and Boland, M. P. 1999. Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants. Animal Science 68: 299-314. Okantah, S.A., Obese, F.Y., Oddoye, E.O.K., Gyawu, P. and Asante, Y. 1999. A Survey on livestock and milk production characteristics of peri-urban agropastoral dairying in Ghana. Journal of Agricultural Science 32:39-46. Okantah, S.A., Obese, F.Y., Oddoye, E.O.K., Kakrikari, P.K., Gyawu, P. and Byant, M. J. 2005. The effect of farm (herd) and season of calving on the Reproductive performance of Sanga cows in smallholder peri-urban dairy farms in the Accra plains. Ghana Journal of Agricultural Science (NARS Edition) 1: 36-42. University of Ghana http://ugspace.ug.edu.gh 74 Opsomer, G., Gröhn, Y.T., Hertl, J., Coryn, M., Deluyker, H. and de Kruif, A. 2000. Risk factors for post-partum ovarian dysfunction in high producing dairy cows in Belgium: a field study. Theriogenology 53: 841–857. Orhue, N. E. J., Nwanze, A.C. and Okafor, A. 2005. Serum total protein, albumin and globulin levels in Trypanosoma brucei-infected rabbits: Effects of orally administered Scoparia dulcis. African Journal of Biotechnology 4(10): 1152-1155. Osafo, E.L.K., Antwi, C., Donkoh, A. and Adu-Dapaah, H. 2013. Feeding graded levels of an improved cultivar of cowpea haulm as supplement for rams fed maize stover diet. International Journal of Agricultural Research 8: 87-93. Osei, S. A., Karikari, P. K., Tuah, A. K., Gyawu, P., Okai, D. B. and Boadu, M. K. 1997. Plasma progesterone measurement as an aid to monitoring the reproductive performance of N’dama cattle. Tropical Agriculture (Trinidad) 74: 294-298. Osei, S. A., Karikari, P. K., Tuah, A. K., Gyawu, P., Opoku, R. S., Asiamah, M. and Heathcote, D. C. 1993. Studies on the reproductive performance of indigenous African Livestock. Results of FAO/IAEA/DGIS co-ordinated Research Programme Organised by the Joint FAO/IAEA Division of Nuclear Teacniques in Food and Agriculture Vienna pp 103-112. Osuji, P.O. 1994. The role of legume forages as supplements to low quality roughages – ILRI Experience. Animal Feed Science and Technology 69:27–38. Otto, F., Baggasse, P., Bogin, E., Harun, M. and Vilela, F. 2000: Biochemical blood profile of Angoni cattle in Mozambique. Israel Journal of Veterinary Medicine 55: 1-9. Oxenreider, S.L. and Wagner, W.C. 1971. Effects of lactation and energy intake on postpartum ovarian activity in the cow. Journal of Animal Science 33: 1026-1031. University of Ghana http://ugspace.ug.edu.gh 75 Pambu-Gollah, R., Cronje, P.B. and Casey, N.H. 2000. An evaluation of the use of blood metabolite concentrations as indicators of nutritional status in free-ranging indigenous goats. South Africa Journal of Animal Science 30(2): 115-120. Patton, J., Kenny, D.A., McNamara, S., Mee, J.F., O’Mara, F.P., Diskin, M.G. and Murphy, J.J. 2007. Relationships among milk production, energy balance, plasma analytes, and reproduction in Holstein-Friesian cows. Journal of Dairy Science 90: 649–658. Payne, W. J. A. 1990. An Introduction to Animal Husbandry in the Tropics. 4th edition. Longman Group Limited UK. 146-148. Peeler, E.J., Otte, M. J. and Esslemont, R. J. 1994. Recurrence odds ratios for peri-parturient diseases and reproductive traits of dairy cows. British Veterinary Journal 150: 481-498. Peter, A.T., Vos, P.L.A.M. and Ambrose, D. J. 2009. Postpartum anestrus in dairy cattle. Theriogenology 71: 1333–1342. Pryce, J. E, Coffey, M. P. and Simm, G. 2001. The relationship between body condition score and reproductive performance. Journal of Dairy Science 84: 1508-1515. Quintavalla, F., Bigliardi, E. and Bertoni, P. 2001. Blood biochemical baseline values in the Ostrich (Struthio camelus). Universita degli studi di Parma, Annali della Facolta di Medical Veterinaria. 21: 61-71. Reist, M., Koller, A., Busato, A., Kupfer, U. and Blum, J. W. 2000. First ovulation and ketone body status in the early postpartum period of dairy cows. Theriogenology 54: 685-701. Roberts, A.J., Nugent, R.A., Klindt, J. and Jenkins, T.G. 1997. Circulating insulin-like growth factor I, insulin-like growth factor binding proteins, growth hormone, and resumption of oestrus in post-partum cows subjected to dietary energy restriction. Journal of Animal Science 75: 1909–1917. University of Ghana http://ugspace.ug.edu.gh 76 Robinson, J. J., Ashworth, C. J., Rooke, J. A., Mitchell, L. M. and McEvoy, T. G. 2006. Nutrition and fertility in ruminant livestock. Animal Feed Science and Technology 126: 256-276. Roche, J. F. and Diskin, M. D. 2001. Resumption of reproductive activity in the early postpartum period of cows. Occasional Publication, British Society of Animal Science 26:31-42. Roche, J.R., Friggens, N.C., Kay, J.K., Fisher, M.W., Stafford, K.J. and Berry, D.P. 2009. Invited review: body condition score and its association with dairy cow productivity, health, and welfare. Journal of Dairy Science 92: 5769–5801. Roche, J.R., Macdonald, K.A., Burke, C.R., Lee, J.M. and Berry, D.P. 2007. Associations among body condition score, body weight and reproductive performance in seasonal-calving dairy cattle. Journal of Dairy Science 90: 376–391. Saleh, N., Mahmud, E. and Waded, E. 2011. Interactions between insulin like growth factor 1, thyroid hormones and blood energy metabolites in cattle with postpartum inactive ovaries. Natural Science 9 (5): 56-63. Samadi, F., Phillips N.J., Blache, D., Martin, G.B. and D’Occhio, M. J. 2013. Interrelationships of nutrition, metabolic hormones and resumption of ovulation in multiparous suckled beef cows on subtropical pastures. Animal Reproduction Science 137: 137-144. Sampson, E.J., Baird, M.A., Burtis, C.A., Smith, E.M., Witte, D.L. and Bayse, D.D. 1980. Coupled-enzyme equilibrium method for measuring urea in serum: optimization and evaluation of the AACC study group on urea candidate reference method. Clinical Chemistry 26: 816-826. SAS, 2003. SAS/STAT User‘s Guide. SAS Institute International, Cary, NC, USA. University of Ghana http://ugspace.ug.edu.gh 77 Savio, J.D., Keenan, L., Boland, M.P. and Roche, J.F. 1990. Pattern of growth of dominant follicles during the oestrous cycle of heifers. Journal of Reproduction and Fertility 83: 663–671. Scaramuzzi, R. J., Baird, D. T., Campbell, B. K., Driancourt, M. A., Dupont, J., Fortune, J. E., Gilchrist, R. B., Martin, G. B., McNatty, K. P., McNeilly, A. S., Monniaux, D., Vinoles, C. and Webb, R. 2011. Regulation of folliculogenesis and the determination of ovulation rate in ruminants. Reproduction Fertility and Development 23: 444-467. Schneider, A., Pfeifer, L.F.M., Hax, L. T., Paludo, G.R., Del Pino, F.A.P., Nelson J. L., Dionello, N.J. L. and Correa, M. N. 2010. Insulin-like growth factor and growth hormone receptor in postpartum lactating beef cows. Persquisa Agropecueria Brasileira 45(8): 925- 931. Schwalm, J. W. and Schultz, L. H. 1976. Relationship of insulin concentration to blood metabolites in the dairy cow. Journal of Dairy Science 59 (2):255-61. Sheldon, I.M., Price, S.B., Cronin, J., Gilbert, R.O. and Gadsby, J. E. 2009. Mechanisms of infertility associated with clinical and subclinical endometritis in high producing dairy cattle. Reproduction in Domestic Animals 44 (Supplement 3): 1–9. Short, R.E., Bellows, R.A., Staigmiller, R.B., Berardinelli, J.G. and Custer, E. E. 1990. Physiological mechanisms controlling anestrus and fertility in postpartum beef cattle. Journal of Dairy Science 68: 799-816. Shrestha, H.K., Nakao, T., Suzuki, T., Akita, M. and Higaki, T. 2005. Relationships between body condition score, body weight, and some nutritional parameters in plasma and University of Ghana http://ugspace.ug.edu.gh 78 resumption of ovarian cyclicity postpartum during pre-service period in high-producing dairy cows in a subtropical region in Japan. Theriogenology 64: 855-866. Shrikhande, G. B., Rode, A. M., Pradhan, M. S. and Satpute, A. K. 2008. Seasonal effects on the composition of blood in cattle. Veterinary World 1(11): 341-342. Sinclair, K.D., Revilla, R., Roche, J.F., Quintans, G., Sanz, A., Mackey, D.R. and Diskin, M.G. 2002. Ovulation of the first dominant follicle arising after day 21 postpartum in suckling beef cows. Journal of Dairy Science 75: 115–126. Smeaton, D. C., McCall, D. G. and Clayton, J. B. 1986. Calving date effects on beef cow productivity. Proceedings of New Zealand Society of Animal Production 46:149. Snijders, S.E.M., Dillon, P., O’Callaghan, D. and Boland, M.P. 2000. Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows. Theriogenology 53: 981–989. Soca, P., Carriquiry, M., Calaramunt, M., Gestido, V. and Meikle, A. 2014. Metabolic and endocrine profiles of primiparous beef cows grazing native grassland. 1. Relationship between body condition score at calving and metabolic profiles during the transition period. Animal Production Science 54: 856-861. Spicer, L.J. 2003. The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals. Domestic Animal Endocrinology 12: 223–245. SPSS Inc 2007: SPSS for windows version 16.0. Chicago, Illinois, U.S. A. Stagg, K., Diskin, M.G., Sreenan, J. M. and Roche, J. F. 1995. Follicular development in long- term anoestrous suckler beef cows fed two levels of energy postpartum. Animal Reproduction Science 38: 49-61. Stagg, K., Spicer, L.J., Sreenan, J.M., Roche, J.F. and Diskin, M.G. 1998. Effect of calf isolation on follicular wave dynamics, gonadotrophin and metabolic hormone changes, and University of Ghana http://ugspace.ug.edu.gh 79 interval to first ovulation in beef cows fed either of two energy levels postpartum. Biology in Reproduction 59: 777–783. Stewart, R.E., Spicer, L.J., Hamilton, T.D. and Keefer, B.E. 1995. Effects of insulin-like growth factor I and insulin on proliferation, and on basal and luteinizing hormone-induced steroidogenesis of bovine thecal cells: involvement of glucose and receptors for insulin- like growth factor I and luteinizing hormone. Journal of Dairy Science 73: 3719–3731. Tamadon, A., Kafi, M., Saeb, M., Mirzaei, A. and Saeb, S. 2011. Relationships between insulin- like growth factor-I, milk yield, body condition score, and postpartum luteal activity in high-producing dairy cows. Tropical Animal Health and Production 43: 29-34. Tanaka, T., Arai, M., Ohtani, S., Uemura, S., Kuroiwa, T., Kim, S. and Kamomae, H. 2008. Influence of parity on follicular dynamics and resumption of ovarian cycle in postpartum dairy cows. Animal Reproduction Science 108: 134–143. Taylor, V.J., Beever, D.E., Bryant, M.J. and Wathes, D.C. 2003. Metabolic profiles and progesterone cycles in first lactation dairy cows. Theriogenology 59: 1661-1677. Tegegne, A., Entwistle, K. W. and Mukasa-Mugerwa. 1992. Nutritional influence on growth and onset of puberty in Boran and Friesian Bulls in Ethiopia. Theriogenology 37:1004-1016. The Merck Veterinary Manual. 2010. A Handbook of diagnosis, therapy, and disease prevention and control for the veterinarian, 10th edn. Kahn CM, Line S (eds), Merck and Co. Inc., New Jersey, USA, pp 905–908. Tindal, J.S., Knaggs, G.S., Hart, I.C. and Blake, L.A. 1978. Release of growth hormone in lactating and non-lactating goats in relation to behaviour, stages of sleep, electroencephalographs, environmental stimuli and levels of prolactin, insulin, glucose and free fatty acids in the circulation. Journal of Endocrinology 76: 333–346. University of Ghana http://ugspace.ug.edu.gh 80 Trinder, P. 1969. Determination of glucose in blood using glucose oxidase with an alternative oxygen receptor. Annals in Clinical Biochemistry 6: 24-27. Velazques, M. A., Spicer, L. J. and Wathes, D. C. 2008. The role of endocrine insulin-like growth factor-I (IGF-I) in female ovine reproduction. Domestic Animal Endocrinology 35: 325-342. Walsh, R.B., Walton, J.S., Kelton, D.F., LeBlanc, S.J., Leslie, K.E. and Duffield, T.F. 2007. The effect of subclinical ketosis in early lactation on reproductive performance of postpartum dairy cows. Journal of Dairy Science 90: 2788-2796. Walsh, S.W., Williams, E.J. and Evans, A.C.O. 2011. A review of the causes of poor fertility in high milk producing dairy cows. Animal Reproduction Science 123: 127-138. Wathes, D.C., Chen, Z., Bourne, N., Taylor, V.J., Coffey, M.P. and Brotherstone, S. 2007. Differences between primiparous and multiparous dairy cows and the inter-relationships between metabolic traits, milk yield and body condition score in the periparturient period. Domestic Animal Endocrinology 33: 203–225. Wettemann, R. P. and Bossis I. 2000. Energy intake regulates ovarian function in beaf cattle. Journal of Animal Science 77 (E. suppl) E: 1-10. Wettemann, R.P., Lents, C.A., Ciccioli, N.H., White, F.J. and Rubio, I. 2003. Nutritional- and suckling-mediated anovulation in beef cows. Journal of Dairy Science 81 (E. Supplement 2): E48–E59. Whittet, K.M., Klopfenstein, T.J., Erickson, G.E., Loy T.W. and McDonald, R.A. 2004. Effect of age, pregnancy, and diet on urinary creatinine excretion in heifers and cows. Nebraska Beef Cattle Reports. Paper 212. University of Ghana http://ugspace.ug.edu.gh 81 Williams. G. L. 1990. Suckling as a regulator of postpartum rebreeding in cattle: A review. Journal of Animal Science 68:831-852. Williams, G. L., Amstalden, M., Garcia, M., Stanko, R. L., Nizielski, S. E., Morrison, C. D. and Keilser, D. H. 2002. Leptin and its role in the central regulation of reproduction in cattle. Domestic Animal Endocrinology 23: 339-349. Williams, E.J., Fischer, D.P., Noakes, D. E., England, G.C.W., Rycroft, A., Dobson, H. and Sheldon, I.M. 2007. The relationship between uterine pathogen growth density and ovarian function in postpartum dairy cows. Theriogenology 68: 549-559. Williams, G.L., Gazal, O.S., Guzmán Vega, G.A. and Stanko, R.L. 1996. Mechanisms regulating suckling mediated anovulation in the cow. Animal Reproduction Science 42: 289–297. Yavas, Y. and Walton, J. S. 2000. Postpartum acyclicity in suckled beef cows: a review. Theriogenology 54: 25-55. Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L. and Friedman, J.M. 1994. Positional cloning of the mouse obese gene and its human homologue. Nature 372: 425- 432. Zulu, V.C., Sawamukai, Y., Nakada, K., Kida, K. and Moriyoshi, M. 2002. Relationships among insulin-like growth factor-1, blood metabolites and post-partum ovarian function in dairy cows. Journal of Veterinary Medical Science 64: 879–885. University of Ghana http://ugspace.ug.edu.gh 82 APPENDICES Appendix 1: Analysis of variance (Type III SS) for milk yield, body weight, and body condition score in Friesian-Sanga crossbred cows 1.1 Partial Milk Yield Source of Variation d.f S.S M.S F-Value Pr > F WK 9 9.64242857 1.07138095 7.56 <0.0001 CAT 2 0.16877083 0.08438542 0.60 0.5530 CAT*WK 18 1.55143750 0.08619097 0.61 0.8883 WK- week; CAT - category of ovarian cyclicity; WK*CAT – ovarian cyclicity and week interaction. 1.2 Body weight Source of Variation d.f S.S M.S F-Value Pr > F MONTH 2 133.150794 66.575397 0.06 0.9403 CAT 2 3858.472222 1929.236111 1.79 0.1810 CAT*MONTH 4 514.652778 128.663194 0.12 0.9749 CAT - category of ovarian cyclicity; CAT*Month – ovarian cyclicity and Month interaction. University of Ghana http://ugspace.ug.edu.gh 83 1.3 BCS Source d.f S.S M.S F Value Pr > F WK 12 2.61 0.22 0.30 0.9882 CAT 2 7.87 3.93 5.49 0.0049 CAT*WK 24 2.09 0.09 0.12 1.0000 CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction Appendix 2: Analysis of variance (Type III SS) for metabolic hormones in Friesian-Sanga crossbred cows 2.1 IGF-I Source d.f S.S M.S F Value Pr > F WK 9 407.703808 45.300423 1.46 0.1695 CAT 2 1853.701954 926.850977 29.93 <0.0001 CAT*WK 18 319.440290 17.746683 0.57 0.9128 CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh 84 2.2 INSULIN Source d.f S.S M.S F Value Pr > F WK 9 2.61085477 0.29009497 0.15 0.9979 CAT 2 6.53074598 3.26537299 1.69 0.1884 CAT*WK 18 10.45489471 0.58082748 0.30 0.9975 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction 2.3 GH Source d.f S.S M.S F Value Pr > F WK 9 7.38885730 0.82098414 0.38 0.9447 CAT 2 0.58896154 0.29448077 0.13 0.8741 CAT*WK 18 23.39965872 1.29998104 0.59 0.8973 ____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh 85 Appendix 3: Analysis of variance (Type III SS) for nutritional metabolites in Friesian-Sanga crossbred cows 3.1 GLUCOSE Source d.f S.S M.S F-Value Pr > F WK 4 1.16714376 0.29178594 1.16 0.3399 CAT 2 0.33518739 0.16759369 0.66 0.5187 CAT*WK 8 1.82645168 0.22830646 0.90 0.5191 CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction 3.2 CHOLESTEROL Source d.f S.S M.S F Value Pr > F WK 4 11.36861145 2.84215286 8.11 <0.0001 CAT 2 4.42862680 2.21431340 6.32 0.0034 CAT*WK 8 2.11736341 0.26467043 0.76 0.6430 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh 86 3.3 TOTAL PROTEIN Source d.f S.S M.S F-Value Pr > F WK 4 2234.376758 558.594189 3.95 0.0068 CAT 2 619.288245 309.644123 2.19 0.1212 CAT*WK 8 1149.661561 143.707695 1.02 0.4339 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction 3.4 ALBUMIN Source d.f S.S M.S F- Value Pr > F WK 4 3019.907288 754.976822 7.12 0.0001 CAT 2 686.945355 343.472678 3.24 0.0467 CAT*WK 8 671.686331 83.960791 0.79 0.6122 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh 87 3.5 GLOBULIN Source d.f S.S M.S F- Value Pr > F WK 4 6944.296918 1736.074230 8.53 <0.0001 CAT 2 1042.158688 521.079344 2.56 0.0862 CAT*WK 8 966.987788 120.873473 0.59 0.7786 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction 3.6 UREA Source d.f S.S M.S F-Value Pr > F WK 4 4.51573874 1.12893469 0.11 0.9772 CAT 2 2.99173794 1.49586897 0.15 0.8604 CAT*WK 8 99.29824388 12.41228049 1.25 0.2876 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh 88 3.7 CREATININE Source d.f S.S M.S F-Value Pr > F WK 4 4756.17082 1189.04271 1.81 0.1389 CAT 2 1547.41252 773.70626 1.18 0.3146 CAT*WK 8 10117.67691 1264.70961 1.93 0.0733 _____________________________________________________________________________________ CAT – category of ovarian cyclicity; WK – week; CAT*WK – Ovarian cyclicity and week interaction University of Ghana http://ugspace.ug.edu.gh