The intent of this chapter is to provide a morphological foundation in the normal cellular process of bovine gamete development so that abnormalities occurring are recognizable. The knowledge gained here is essential to begin to understand the significance of many of the common bull sperm abnormalities encountered in the clinics. Spermatogenesis is divided into three phases (i. e., Mitosis, Meiosis and Spermiogenesis) all happening in the seminiferous epithelium. The 'Cycle of the Bovine Seminiferous Epithelium' is explained in relation to these phases. Information is provided as to how to identify the stages of the bovine cycle and the steps of spermiogenesis at the histological and ultrastructural levels in preparation to recognize where and when in the cycle a spermatid abnormality arises. Spermiogenesis, the last phase of spermatogenesis, is the most revealing phase to recognize gamete abnormalities as this is where spermatid head and tail differentiation take place and spermatid compartments materialize. The formation of the nucleus, acrosome, manchette, perinuclear theca, axoneme, outer dense fibers, fibrous sheath, connecting piece and mitochondrial sheath occur during this phase and are evaluated. The origins and assembly of a number of essential proteins compartmentalizing the sperm head and tail as well as defects arising during spermiogenesis are reviewed.

Nulliparous yearling beef heifers (n=360) were used to evaluate the effects of maternal dietary protein during the periconception and first trimester periods of gestation on postnatal growth, feedlot performance, carcass characteristics, and the expression of genes associated with appetite in the arcuate nucleus of their male progeny. Heifers were individually fed a diet of 1.18g crude protein (CP)/day High protein (HPeri) or 0.62g CP/day Low protein (LPeri) beginning 60days before conception. From 24 to 98days post-conception (dpc), half of each treatment group changed to the alternative post-conception diet and were fed 1.49g CP/day (HPost) or 0.88g CP/day (LPost) yielding four treatment groups in a 2×2 factorial design. From day 98 of gestation, heifers received a common diet until parturition. Calves were weaned at 183days and developed on pasture before feedlot entry. Bulls underwent a 70-day Residual Feed Intake (RFI) feedlot test commencing at 528days of age. Feedlot entry and final body weight (BW), feedlot average daily gain (ADG) and RFI were not different (p>0.05). Progeny of dams that had a change in diet (LPeri/HPost and HPeri/LPost) had 9% higher daily dry matter intake (DMI) during the RFI test (p<0.05) than progeny of dams that received low diet throughout both the peri-conception period and first trimester (LPeri/LPost). Further, mRNA expression of the appetite-stimulating agouti-related protein (AGRP) was increased in the arcuate nucleus of High Peri/LPost bulls (p<0.05). Longissimus dorsi muscle cross sectional area, carcass dressing percentage, and estimated retail beef yield (RBY) were all higher (p<0.05), and rump (P8) fat tended to be lower (p=0.07), for bulls from HPost dams despite no difference in carcass weight (p<0.05). This study is of commercial importance to the livestock industry as specific periods of maternal dietary supplementation may increase feed intake, enhance progeny muscling, and alter fat deposition leading to improvement in efficiency of meat production in beef cattle.

The lack of standardization in the laboratory assessment of semen questions the reliability of semen analysis, and makes meaningful interpretation of these evaluations impossible. We herein describe a standardization program for morphology assessment currently in place in Australia and outline the methods used, both for the categorization of the abnormalities, including newly described abnormalities, and those that permit standardized microscopic assessment between laboratories.

This study evaluated the effect of protein restriction during the periconception (PERI) and first trimester (POST) periods on maternal performance, physiology and early fetal growth. Yearling nulliparous heifers (n=360) were individually fed a diet high or low in protein (HPeri and LPeri respectively) beginning 60 days before conception. From 24 to 98 days post-conception (dpc), half of each treatment group changed to the alternative post-conception high- or low-protein diet (HPost and LPost respectively), yielding four groups in a 2×2 factorial design with a common diet until parturition. Protein restriction was associated with lower bodyweight subsequent to reduced (but positive) average daily weight gain (ADG) during the PERI and POST periods. During the POST period, ADG was greater in LPeri than HPeri heifers and tended to be greater in LPost than HPost heifers during the second and third trimester. Bodyweight was similar at term. The pregnancy rate did not differ, but embryo loss between 23 and 36 dpc tended to be greater in LPeri than HPeri heifers. Overall, a greater proportion of male fetuses was detected (at 60 dpc 63.3% male vs 36.7% female). Protein restriction altered maternal plasma urea, non-esterified fatty acids, progesterone, leptin and insulin-like growth factor 1 at critical stages of fetal development. However, profiles varied depending on the sex of the conceptus.

Few studies have investigated the effects of nutrition during the periconception and early gestation periods on fetal and placental development in cattle. In this study, nulliparous yearling heifers (n=360) were individually fed a diet high or low in protein (HPeri and LPeri) beginning 60 days before conception. From 24 to 98 days after conception, half of each treatment group was changed to the alternative high- or low-protein diet (HPost and LPost) yielding four groups in a 2×2 factorial design. A subset of heifers (n=46) was necropsied at 98 days after conception and fetoplacental development assessed. Placentome number and volume decreased in response to LPeri and LPost diets respectively. Absolute lung, pancreas, septum and ventricle weights decreased in LPost versus HPost fetuses, whereas the post-conception diet altered absolute and relative liver and brain weights depending on sex. Similarly, changes in fetal hepatic gene expression of factors regulating growth, glucose output and lipid metabolism were induced by protein restriction in a sex-specific manner. At term, neonatal calf and placental measures were not different. Protein restriction of heifers during the periconception and early gestation periods alters fetoplacental development and hepatic gene expression. These changes may contribute to functional consequences for progeny, but this may not be apparent from gross morphometry at birth.

This experiment evaluated the effect of pre-weaning plane of nutrition of dams upon reproductive development in Bos indicus x Bos taurus bull offspring reared under extensively managed conditions in the northern Australia rangelands. Following artificial insemination (AI cohort; n = 26), or natural mating (NM cohort; n = 36), grazing primiparous heifers received either nil supplement (Control; CON), thereby experiencing a moderate nutritional restriction, or were provided a protein supplement (SUPP) between parturition and weaning at mean age 199 ± (SD) 26 d. Bull progeny grazed rangeland pastures without supplementation from weaning until the experiment concluded at 675 d. At 120 ± 3 d and 140 d ± 10 d age, within the AI and NM cohort, respectively, bull calves were subjected to a GnRH challenge (1.5 μg/kg of body weight i.m.). Jugular blood samples collected immediately before and at 60 min after administration of GnRH were analysed for LH, FSH, testosterone and inhibin concentrations. Overall mean concentrations of testosterone in SUPP bulls were greater in both the AI cohort (P = 0.05) and the NM cohort (P = 0.06). At 60 d intervals during the post-weaning period, scrotal circumference (SC) was measured and semen collected to assess concentration, progressive motility and morphology of sperm. Bulls reared by SUPP heifers had greater (P = 0.05) SC at 375 d and tended to have greater (P = 0.09) mean percentage of morphologically normal sperm (PNS). Within the NM cohort, NMSUPP bulls had greater (P = 0.04) overall mean SC and tended (P = 0.07) to demonstrate both greater progressive motility and PNS. A greater incidence of sperm morphological abnormalities, associated with sexual immaturity, were observed in CON bulls. Consequently, NMCON bulls demonstrated delayed (P = 0.03) age of sexual maturity as compared to NMSUPP bulls. In summary, improving the plane of nutrition supplied to Bos indicus x Bos taurus bulls between parturition and weaning via moderate supplementation of grazing dams reduces age at sexual maturity with consequent economic advantages to the producer.

The practice of feeding replacement-breeding bulls on high energy diets after weaning to meet liveweight (LW) and carcass expectations between 18 and 24 months of age negatively affects reproductive potential. This experiment reports upon the effects of an alternative management strategy aimed at improving calfhood nutrition in rangeland-reared bulls to enhance LW and live carcass characteristics at 2 years. Following artificial insemination (AI cohort; n = 26), or natural mating, subsequent to the addition of bulls at 39 d post-AI (NM cohort; n = 36), primiparous Santa Gertrudis heifers grazing rangeland pastures with bull calf progeny were allocated at parturition to receive either nil supplement (control; CON) or provided with unrestricted access to a pelleted vegetable protein meal-based supplement containing 35% CP (SUPP) until weaning at 199 ± (SD) 26 d. The mean estimated pellet consumption by SUPP heifers during lactation was 2.6 ± (SEM) 0.5 kg DM daily. Grazing diet quality measurements indicated nutritional restriction of CON heifers until at least 115 d of lactation. This was confirmed by lower blood urea nitrogen concentrations at 88 d (P < 0.001) and greater mean NEFA (P < 0.001) concentrations. Rainfall during mid-lactation subsequently improved grazing diet quality; thus the CON heifers experienced moderate nutritional restriction across lactation, but sufficient to reduce milk yield by 1.6 kg/d (P < 0.001) and maternal LW at weaning by 18.4 kg (P < 0.001). Bulls reared by SUPP heifers were 17.5 kg heavier at weaning (P = 0.001) and had elevated IGF-I and leptin concentrations between 4 and 4.5 months of age (P < 0.05). Effects on metabolic hormones during calfhood were cohort specific, with greater concentrations of IGF-I confined to AISUPP bulls and NMSUPP bulls demonstrating greater concentrations of leptin. Bulls were amalgamated at weaning and grazed common pastures without supplementation until the experiment concluded at 675 d. Pre-weaning plane of nutrition did not affect the LW, carcass fat depth, IGF-I or leptin concentrations of bulls after weaning. Mean eye muscle area (EMA) was greater in SUPP compared to CON bulls (68.5 ± 0.9 cm2vs 65.2 ± 0.9 cm2; P < 0.05) and AISUPP bulls tended to have greater EMA (P = 0.06) than AICON bulls from 495 d of age. Thus when primiparous heifers experience moderate nutritional restriction during lactation, supplementation may have persistent effects upon increasing carcass muscle characteristics of bull progeny.

During ovarian development stroma from the mesonephros penetrates and expands into the ovarian primordium and thus appears to be involved, at least physically, in the formation of ovigerous cords, follicles and surface epithelium. Cortical stromal development during gestation in bovine fetal ovaries (n=27) was characterised by immunohistochemistry and by mRNA analyses. Stroma was identified by immunostaining of stromal matrix collagen type I and proliferating cells were identified by Ki67 expression. The cortical and medullar volume expanded across gestation, with the rate of cortical expansion slowing over time. During gestation, the proportion of stroma in the cortex and total volume in the cortex significantly increased (P<0.05). The proliferation index and numerical density of proliferating cells in the stroma significantly decreased (P<0.05), whereas the numerical density of cells in the stroma did not change (P>0.05). The expression levels of 12 genes out of 18 examined, including osteoglycin (OGN) and lumican (LUM), were significantly increased later in development (P<0.05) and the expression of many genes was positively correlated with other genes and with gestational age. Thus, the rate of cortical stromal expansion peaked in early gestation due to cell proliferation, whilst late in development expression of extracellular matrix genes increased.

Movement and expansion of mesonephric-derived stroma appears to be very important in the development of the ovary. Here, we examined the expression of 24 genes associated with stroma in fetal ovaries during gestation (n = 17; days 58–274) from Bos taurus cattle. RNA was isolated from ovaries for quantitative RT-PCR. Expression of the majority of genes in TGFβ signalling, stromal transcription factors (NR2F2, AR), and some stromal matrix genes (COL1A1, COL3A1 and FBN1, but not FBN3) showed a positive linear increase with gestational age. Expression of genes associated with follicles (INSL3, CYP17A1, CYP11A1 and HSD3B1), was low until mid-gestation and then increased with gestational age. LHCGR showed an unusual bimodal pattern; high levels in the first and last trimesters. RARRES1 and IGFBP3 also increased with gestational age. To relate changes in gene expression in stromal cells with that in non stromal cells during development of the ovary we combined the data on the stromal genes with another 20 genes from non stromal cells published previously and then performed hierarchical clustering analysis. Three major clusters were identified. Cluster 1 genes (GATA4, FBN3, LHCGR, CYP19A1, ESR2, OCT4, DSG2, TGFB1, CCND2, LGR5, NR5A1) were characterised by high expression only in the first trimester. Cluster 2 genes (FSHR, INSL3, HSD3B1, CYP11A1, CYP17A1, AMH, IGFBP3, INHBA) were highly expressed in the third trimester and largely associated with follicle function. Cluster 3 (COL1A1, COL3A1, FBN1, TGFB2 TGFB3, TGFBR2, TGFBR3, LTBP2, LTBP3, LTBP4, TGFB1I1, ALDH1A1, AR, ESR1, NR2F2) had much low expression in the first trimester rising in the second trimester and remaining at that level during the third trimester. Cluster 3 contained members of two pathways, androgen and TGFβ signalling, including a common member of both pathways namely the androgen receptor cofactor TGFβ1 induced transcript 1 protein (TGFB1I1; hic5). GATA4, FBN3 and LHCGR, were highly correlated with each other and were expressed highly in the first trimester during stromal expansion before follicle formation, suggesting that this could be a critical phase in the development of the ovarian stroma.

The greatest loss in ruminant production systems occurs during the neonatal period. The maternal environment (nutrition and physiologic status) influences neonatal mortality and morbidity as it reportedly affects (a) Dystocia, both via increasing birth weight and placental dysfunction; (b) Neonatal thermoregulation, both via altering the amount of brown adipose tissue and its ability to function via effects upon the hypothalamic-pituitary-thyroid axis; (c) Modification of the developing immune system and its symbiotic nutrient sources; (d) Modification of maternal and neonatal behavior.

Maternal low protein diets prior to conception alter embryo and fetal development and are associated with detrimental outcomes in the offspring in many species. The aim of this study in beef cattle was to investigate the effect of preconception dietary protein upon maternal and offspring behaviour at birth concomitant with the associated hormonal profile. Sixty days prior to conception, nulliparous yearling heifers (n = 85) were fed either a High (PreH: 18%; n = 43) or Low (PreL: 10%; n = 42) crude protein diet, followed by a control diet throughout gestation. After calving, each cow-calf pair was penned individually, accelerometers fitted, and each pen observed continuously via video recordings. Cows fed on the low protein diet during preconception showed an increase in standing time (P < 0.01); while calves born to heifers receiving the PreL diet showed an increase in suckling time (P = 0.04). These calves were also heavier at birth than calves from PreH mothers (P < 0.01). In conclusion, low maternal dietary protein prior to conception in beef heifers modifies both offspring feeding behaviour and birth weight, and cow's standing times.

This study reports the distribution of sperm morphology defects by breed, age, season and region of 11,387 bulls in 500 herds in Australia and near Pacific Islands during annual BBSE. Bull location was divided into 4 broad climatic regions based upon temperature, vegetation and climatic risk. Taking into account the impact of age, season, region, and breed there were differences between breeds in both percent morphologically normal sperm and in some individual categories of sperm abnormality (P < 0.001). Independent of breed, season and region, proximal droplets were significantly increased in bulls less than 20 months of age. This is the first study to comprehensively collect data from this wide geographical area and compare sperm morphology profiles among the Bos indicus and Bos taurus breeds. The findings of this study will act as a guide for veterinary practitioners and cattle breeders in the proportion of bulls that can be expected to pass the PNS test, by breed, age and region, based on a robust data set.

Cells on the surface of the mesonephros give rise to replicating Gonadal Ridge Epithelial-Like (GREL) cells, the first somatic cells of the gonadal ridge. Later germ cells associate with the GREL cells in the ovigerous cords, and the GREL cells subsequently give rise to the granulosa cells in follicles. To examine these events further, 27 bovine fetal ovaries of different gestational ages were collected and prepared for immunohistochemical localisation of collagen type I and Ki67 to identify regions of the ovary and cell proliferation, respectively. The non-stromal cortical areas (collagen-negative) containing GREL cells and germ cells and later in development, the follicles with oocytes and granulosa cells, were analysed morphometrically. Another set of ovaries (n = 17) were collected and the expression of genes associated with germ cell lineages and GREL/granulosa cells were quantitated by RT-PCR. The total volume of non-stromal areas in the cortex increased significantly and progressively with ovarian development, plateauing at the time the surface epithelium developed. However, the proportion of non-stromal areas in the cortex declined significantly and progressively throughout gestation, largely due to a cessation in growth of the non-stroma cells and the continued growth of stroma. The proliferation index in the non-stromal area was very high initially and then declined substantially at the time follicles formed. Thereafter, it remained low. The numerical density of the non-stromal cells was relatively constant throughout ovarian development. The expression levels of a number of genes across gestation either increased (AMH, FSHR, ESR1, INHBA), declined (CYP19A1, ESR2, ALDH1A1, DSG2, OCT4, LGR5) or showed no particular pattern (CCND2, CTNNB1, DAZL, FOXL2, GATA4, IGFBP3, KRT19, NR5A1, RARRES1, VASA, WNT2B). Many of the genes whose expression changed across gestation, were positively or negatively correlated with each other. The relationships between these genes may reflect their roles in the important events such as the transition of ovigerous cords to follicles, oogonia to oocytes or GREL cells to granulosa cells.

Brown adipose tissue (BAT) undergoes pronounced changes after birth coincident with the loss of the BAT-specific uncoupling protein (UCP)1 and rapid fat growth. The extent to which this adaptation may vary between anatomical locations remains unknown, or whether the process is sensitive to maternal dietary supplementation. We, therefore, conducted a data mining based study on the major fat depots (i.e. epicardial, perirenal, sternal (which possess UCP1 at 7 days), subcutaneous and omental) (that do not possess UCP1) of young sheep during the first month of life. Initially we determined what effect adding 3% canola oil to the maternal diet has on mitochondrial protein abundance in those depots which possessed UCP1. This demonstrated that maternal dietary supplementation delayed the loss of mitochondrial proteins, with the amount of cytochrome C actually being increased. Using machine learning algorithms followed by weighted gene co-expression network analysis, we demonstrated that each depot could be segregated into a unique and concise set of modules containing co-expressed genes involved in adipose function. Finally using lipidomic analysis following the maternal dietary intervention, we confirmed the perirenal depot to be most responsive. These insights point at new research avenues for examining interventions to modulate fat development in early life.

Nutritional perturbation during gestation alters male reproductive development in rodents and sheep. In cattle both the developmental trajectory of the feto-placental unit and its response to dietary perturbations is dissimilar to that of these species. This study examined the effects of dietary protein perturbation during the peri-conception and first trimester periods upon reproductive development in bulls. Nulliparous heifers (n=360) were individually fed a high- or low-protein diet (HPeri and LPeri) from 60 days before conception. From 24 until 98 days post conception, half of each treatment group changed to the alternative post-conception high- or low-protein diet (HPost and LPost) yielding four treatment groups in a 2×2 factorial design. A subset of male fetuses (n=25) was excised at 98 days post conception and fetal testis development was assessed. Reproductive development of singleton male progeny (n=40) was assessed until slaughter at 598 days of age, when adult testicular cytology was evaluated. Low peri-conception diet delayed reproductive development: sperm quality was lowered during pubertal development with a concomitant delay in reaching puberty. These effects were subsequent to lower FSH concentrations at 330 and 438 days of age. In the fetus, the low peri-conception diet increased the proportion of seminiferous tubules and decreased blood vessel area in the testis, whereas low first trimester diet increased blood vessel number in the adult testis. We conclude that maternal dietary protein perturbation during conception and early gestation may alter male testis development and delay puberty in bulls.

Genetically similar nulliparous Polled Hereford heifers from a closed pedigree herd were used to evaluate the effects of dietary protein during the first and second trimester of gestation upon foetal, placental and postnatal growth. Heifers were randomly allocated into two groups at 35 days after artificial insemination (35 days post conception (dpc)) to a single bull and fed high (15.7% CP) or low (5.9% CP) protein in the first trimester (T1). At 90 dpc, half of each nutritional treatment group changed to a high- or low-protein diet for the second trimester until 180 dpc (T2). High protein intake in the second trimester increased birth weight in females (P=0.05), but there was no effect of treatment upon birth weight when taken over both sexes. Biparietal diameter was significantly increased by high protein in the second trimester with the effect being greater in the female (P=0.02), but also significant overall (P=0.05). Placental weight was positively correlated with birth weight, fibroblast volume and relative blood vessel volume (P<0.05). Placental fibroblast density was increased and trophoblast volume decreased in the high-protein first trimester treatment group (P<0.05). There was a trend for placental weight to be increased by high protein in the second trimester (P=0.06). Calves from heifers fed the high-protein treatment in the second trimester weighed significantly more on all occasions preweaning (at 1 month (P=0.0004), 2 months (P=0.006), 3 months (P=0.002), 4 months (P=0.01), 5 months (P=0.03), 6 months (P=0.001)), and grew at a faster rate over the 6-month period. By 6 months of age, the calves from heifers fed high nutrition in the second trimester weighed 33 kg heavier than those fed the low diet in the second trimester. These results suggest that dietary protein in early pregnancy alters the development of the bovine placenta and calf growth to weaning.

Brown adipose tissue acting through a unique uncoupling protein (UCP1) has a critical role in preventing hypothermia in newborn sheep but is then thought to rapidly disappear during postnatal life. The extent to which the anatomical location of fat influences postnatal development and thermogenic function in adulthood, particularly following feeding, is unknown, and we examined both in our study. Changes in gene expression of functionally important pathways (i.e., thermogenesis, development, adipogenesis, and metabolism) were compared between sternal and retroperitoneal fat depots together with a representative skeletal muscle over the first month of postnatal life, coincident with the loss of brown fat and the accumulation of white fat. In adult sheep, implanted temperature probes were used to characterize the thermogenic response of fat and muscle to feeding and the effects of reduced or increased adiposity. UCP1 was more abundant in sternal fat than in retroperitoneal fat and was retained only in the sternal depot of adults. Distinct differences in the abundance of gene pathway markers were apparent between tissues, with sternal fat exhibiting some similarities with muscle that were not apparent in the retroperitoneal depot. In adults, the postprandial rise in temperature was greater and more prolonged in sternal fat than in retroperitoneal fat and muscle, a difference that was maintained with altered adiposity. In conclusion, sternal adipose tissue retains UCP1 into adulthood, when it shows a greater thermogenic response to feeding than do muscle and retroperitoneal fat. Sternal fat may be more amenable to targeted interventions that promote thermogenesis in large mammals.

Breeding bulls are commonly fed high-energy diets, which may induce subacute ruminal acidosis (SARA). In this experiment, 8 Santa Gertrudis bulls (age 20 ± 6 mo) were used to evaluate the extent and duration of effects of SARA on semen quality and the associated changes in circulating hormones and metabolites. The bulls were relocated and fed in yards with unrestricted access to hay and daily individual concentrate feeding for 125 d before SARA challenge. Semen was collected and assessed at 14-d intervals before the challenge to ensure acclimatization and the attainment of a stable spermiogram. The challenge treatments consisted of either a single oral dose of oligofructose (OFF; 6.5 g/kg BW) or an equivalent sham dose of water (Control). Locomotion, behavior, respiratory rate, and cardiovascular and gastrointestinal function were intensively monitored during the 24-h challenge period. Rumen fluid samples were retained for VFA, ammonia, and lactate analysis. After the challenge, semen was then collected every third day for a period of 7 wk and then once weekly until 12 wk, with associated blood collection for FSH, testosterone, inhibin, and cortisol assay. Percent normal sperm decreased in bulls dosed with OFF after the challenge period ( < 0.05) and continued to remain lower on completion of the study at 88 d after challenge. There was a corresponding increase in sperm defects commencing from 16 d after challenge. These included proximal cytoplasmic droplets ( < 0.001), distal reflex midpieces ( = 0.01), and vacuole and teratoid heads ( < 0.001). Changes in semen quality after challenge were associated with lower serum testosterone ( < 0.001) and FSH ( < 0.05). Serum cortisol in OFF bulls tended to be greater ( = 0.07) at 7 d after challenge. This study shows that SARA challenge causes a reduction in sperm quality sufficient to preclude bulls from sale as single sire breeding animals 3 mo after the event occurred.