J.M. Sreenan

Effects of nutrition and metabolic status on circulating hormones and ovarian follicle development in cattle

Nutrition is a major factor affecting cow reproductive efficiency. Long-term moderate or chronic dietary restriction results in a gradual reduction in dominant follicle (DF) growth rate, maximum diameter and persistence. Animals become anoestrus when they lose on average 22-24% of their initial body weight. There is evidence of significant animal-to-animal variation in the interval from the imposition of dietary restriction to onset of anoestrus and from the recommencement of re-alimentation to resumption of ovulation. In contrast, acute dietary restriction to 40% of maintenance requirements rapidly reduces dominant follicle growth rate and maximum diameter and induces anoestrus in a high proportion (60%) of heifers within 13-15 days of dietary restriction. In lactating dairy and beef cows negative energy balance or reduced dietary intake in the early post-partum period, while not affecting the population of small-to-medium size follicles, adversely affects the size and ovulatory fate of the dominant follicle. Re-alimentation of nutritionally induced anoestrous heifers results in an initial gradual increase in dominant follicle growth rate and maximum diameter, followed by a more accelerated increase in dominant follicle growth rate and maximum diameter as the time of resumption of ovulation approaches. Increased dominant follicle growth rate and maximum diameter are associated with increased peripheral concentrations of IGF-I, pulsatile LH and oestradiol. Direct nutritional effects on ovarian function appear to operate through hepatic rather than follicular regulation of IGF-I, and on systemic concentrations of IGF-I BPs and insulin; cumulatively reducing follicular responsiveness to LH and ultimately shutting down follicular oestradiol production. Indirect nutritional effects are apparently mediated through altering the GnRH pulse generator and in-turn selectively reducing pulsatile LH secretion without any apparent adverse effect on FSH secretory patterns. Endogenous opioid peptides, NPY and glucose appear to play a role in the nutritional regulation of GnRH release and in turn pulsatile LH secretion.

Embryo and foetal loss in beef heifers between day 14 of gestation and full term

Following insemination, reproductive failure in cattle is largely manifested as embryo mortality and is a major source of financial loss to livestock producers. Ongoing studies at this laboratory into factors affecting embryo mortality have facilitated the collection of new data on the extent and timing of embryo and foetal mortality in cattle. Oestrus was synchronised in 158 beef cross heifers and following artificial insemination, embryo and foetal survival rates were determined on days 14 and 30 after insemination and subsequently at calving. Embryo survival rates measured on days 14, 30 and at full term were similar at 68%, 76% and 71.8%, respectively (P0.05). Based on morphological examination, all the 14-day-old embryos recovered were assessed as grade 1. These results provide new information indicating that most embryo losses in heifers have occurred before day 14 after insemination.

Follicular development in long-term anoestrous suckler beef cows fed two levels of energy postpartum

Abstract Follicular growth patterns were monitored daily by ultrasonography, from calving until completion of the first ovarian cycle postpartum in beef cows fed either a low (L; n = 11) or high (H; n = 12) energy diet. Mean cow body condition score at calving was 2.1 (range 1.75–2.25). The mean (± standard error of the mean) number of days postpartum to detection of the first follicle wave was 10.4±0.9 and 9.6±0.9 days for the L and H groups respectively (P>0.10). L diet cows tended to have more (P=0.09) medium-dominant follicle waves before first ovulation than H diet cows. Because the first two follicle waves postpartum consisted of the medium follicles, this resulted in a slightly longer (P>0.10) interval to detection of the first dominant follicle wave in the L cows than in the H cows. The total number of follicle waves before first ovulation was 10.6±1.2 anand 6.8±1.2 (P 0.10) but did change (P

Extent, pattern and factors associated with late embryonic loss in dairy cows

Intensive genetic selection for increased milk production, coupled with increased dry matter intakes has led to significant improvements in cow milk yield, however, this increase in milk output has been accompanied by a decline in cow fertility. It has been suggested that there is a higher increment of late embryonic loss in high-yielding than in moderate yielding cows or in heifers. The objectives of this study were to establish the extent and pattern of embryonic loss, from days 28 to 84 of gestation, and to examine possible relationships between cow milk yield, cow genetic merit, parity, calving to insemination interval and embryonic loss in dairy cows managed mainly under pasture-based milk production systems. Multiparous dairy cows (n=1046) located on 8 farms and nulliparous dairy heifers (n=162) located on five of these farms were used in this study. The extent and timing of embryonic loss was measured by ultrasound scanning of the cows and heifers at 14-day intervals between days 28 and 84 of gestation. Positive diagnosis of pregnancy was based on the presence of an embryo or foetus with a visible heartbeat and, at the later scans, visible movement, whose size was compatible with stage of gestation and also on the presence of clear amniotic fluid of the cows and heifers presented as presumed pregnant on day 28 after insemination, 67 and 81%, respectively had a viable embryo. The subsequent embryonic loss rate between days 28 and 84 of gestation was similar (P>0.05) for cows (7.2%) and heifers (6.1%) and the pattern of loss over this period was also similar (P>0.05) for cows and heifers. There was no significant association (P>0.05) between level of milk production or milk energy output measured to day 120 of lactation and embryonic loss rate. Similarly, there was no significant relationship (P>0.05) between % milk fat, % milk protein and % milk lactose and embryonic loss rate. The extent and pattern of embryonic loss were not related (P>0.05) to either cow or to cow sire genetic merit. There was no significant (P>0.05) relationship between the calving to first service interval and embryonic loss. The extent of embryonic loss was greater (P<0.05) in cows that lost body condition between days 28 and 56 of gestation compared with cows than either maintained or improved in body condition.

Effect of level of dietary n-3 polyunsaturated fatty acid supplementation on systemic and tissue fatty acid concentrations and on selected reproductive variables in cattle

Reproductively normal crossbred beef heifers were individually offered a diet of barley straw and concentrate supplemented with one of four levels of a fish oil (FO) enriched supplement. Following oestrous cycle synchronisation, blood samples were collected at appropriate intervals for the measurement of progesterone (P(4)), oestradiol (E(2)), fatty acids, insulin-like growth factor 1 (IGF-1) and metabolites. On days 15 and 16 of the cycle, oxytocin was administered intravenously and the prostaglandin F(2alpha) (PGF(2alpha)) response was measured as venous concentrations of 13,14-dihydro-15-keto PGF(2alpha) (PGFM). The heifers were slaughtered on days 17 or 18 of the oestrous cycle and endometrial tissue, rumen fluid and follicular fluid were collected for determination of fatty acid concentrations. In general there was no effect (P>0.05) of diet on plasma P(4) or E(2) concentrations. Increasing FO supplementation increased CL diameter on day 7 post-oestrus (P<0.0001) but had no effect on diameter on day of slaughter (P>0.05). On day 15, PGFM concentration was greater on the highest level of FO supplementation compared to controls (P<0.05), however, there were no differences between other diet comparisons (P>0.05). There was no effect of diet on PGFM concentration on day 16 (P>0.05). There was a strong positive relationship between plasma and uterine endometrial concentrations of both EPA (R(2)=0.86; P<0.0001) and total n-3 PUFA (R(2)=0.77; P<0.0001). IGF-1 concentrations increased on all diets and were greatest at the highest level of n-3 PUFA supplementation (P<0.05).

Development and validation of a biosensor-based immunoassay for progesterone in bovine milk

We have developed a rapid automated immunoassay, using the BIACORE surface plasmon resonance (SPR) biosensor, to measure progesterone in bovine milk. The assay was designed as an inhibition assay with progesterone covalently immobilised to the carboxymethyl dextran matrix of a CM5 sensor chip. A fixed amount of monoclonal anti-progesterone antibody 39C5H7 was mixed 9:1 with the sample and the amount of free antibody was then determined using biomolecular interaction analysis (BIA) by injection of the mixture over the immobilised progesterone sensor surface. The assay was designed to cover the concentration range 0.5 to 50 ng/ml. The limit of detection (LOD) was 3.56 ng/ml. Reproducibility of the assay was very good with both intra-assay and inter-assay coefficients of variation <5%. As results become available within minutes of injection and the procedure involves fully automated instrumentation, we believe that this BIA assay for progesterone in milk could be used in-line in the milking parlour and, thus, provide an important tool for reproductive management of dairy cattle to detect heat and predict pregnancy.

Dietary n-3 polyunsaturated fatty acids alter the expression of genes involved in prostaglandin biosynthesis in the bovine uterus.

Nutrition plays a critical role in the regulation of cow fertility. There is emerging evidence that dietary long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) may act as specific regulators of some reproductive processes. In vitro studies suggest that the n-3 PUFAs, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) may play pivotal roles by suppressing the synthesis of uterine prostaglandin F(2alpha) (PGF(2alpha)) which is centrally involved in the control of the bovine oestrous cycle and in early embryo survival. The objective of the current study was to determine the effect of dietary inclusion of n-3 PUFA on uterine endometrial mRNA expression of key genes regulating PGF(2alpha) biosynthesis. Beef heifers were fed either a low (CON; n=10) or high (HIGH PUFA; n=10) n-3 PUFA diet for 45 days and endometrial tissues were harvested following slaughter. Following analysis, tissues within each dietary group were ranked on the basis of their PUFA concentrations and the highest (n=7) and lowest (n=7) within each of HIGH PUFA and CON, respectively, were used in gene expression studies. Endometrial n-3 PUFA concentrations were more than two-fold higher (P<0.05) and EPA concentrations alone more than seven-fold higher (P<0.01) in the HIGH PUFA than the CON group. Endometrial concentrations of arachidonic acid, were lower (P<0.001) in the tissues from HIGH PUFA than those from the CON group. Total RNA was isolated from all endometrial tissues and real-time reverse transcription (RT) PCR conducted to compare the relative expression of 11 genes with known involvement in uterine biosynthesis of 2-series prostaglandins. Expression of mRNA for prostaglandin E synthase (PGES) and peroxisome proliferator-activated receptors, PPAR alpha and delta was increased (P<0.05) while mRNA expression of phospholipase A(2) (PLA(2)) was decreased (P=0.06) in the HIGH PUFA endometrial tissues. Expression of genes coding for the oxytocin receptor (OTR), phospholipase C (PLC), cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), PGE(2) 9-ketoreductase (9-KPR), prostaglandin F synthase (PGFS), and the nuclear transcription factor, PPAR gamma was not different (P>0.05) between HIGH PUFA and CON tissues. Overall the results indicate that key genes regulating uterine PGF(2alpha) biosynthesis can be regulated by dietary inclusion of LC n-3 PUFA which may influence uterine function and embryo survival.

Effect of Elevated Systemic Concentrations of Ammonia and Urea on the Metabolite and Ionic Composition of Oviductal Fluid in Cattle

Abstract High dietary protein leads to elevated systemic concentrations of ammonia and urea, and these, in turn, have been associated with reduced fertility in cattle. The effect of elevating systemic concentrations of ammonia and urea on the concentrations of electrolytes and nonelectrolytes in bovine oviductal fluid were studied using estrus-synchronized, nulliparous heifers (n = 25). Heifers were randomly assigned to 1 of 3 treatments consisting of jugular vein infusion with either ammonium chloride (n = 8), urea (n = 8), or saline (n = 9). Oviducts were catheterized, and fluid was recovered over a 3-h period on either Day 2 or 8 of the estrous cycle. No difference (P > 0.05) was found in the concentrations of any electrolyte or nonelectrolyte between oviducts ipsi- or contralateral to the corpus luteum. Plasma and oviductal concentrations of urea were increased by infusion with urea (P < 0.001) and ammonium chloride (P < 0.05) but not by saline (P > 0.05). Plasma and oviductal concentrations of ammonia were elevated by infusion with ammonium chloride (P < 0.001) but not by infusion with urea or saline (P > 0.05). No effect (P > 0.05) of treatment was found on oviductal or plasma concentrations of glucose, lactate, magnesium, potassium, or sodium or on plasma concentrations of insulin or progesterone. The concentration of calcium in oviductal fluid was reduced by urea infusion and was negatively associated with systemic and oviductal concentrations of urea. Oviductal concentrations of sodium were higher on Day 8 than on Day 2 (P < 0.05). No effect of sample day was found on any of the other electrolytes or nonelectrolytes measured (P > 0.05). Elevated systemic concentrations of ammonia and urea are unlikely to reduce embryo survival through disruptions in the oviductal environment.

Effects of changes in the concentration of systemic progesterone on ions, amino acids and energy substrates in cattle oviduct and uterine fluid and blood

Early embryo loss is a major factor affecting the conception rate in cattle. Up to 40% of cattle embryos die within 3 weeks of fertilisation while they are nutritionally dependent on oviduct and uterine fluids for their survival. Inadequate systemic progesterone is one of the factors contributing to this loss. We have characterised the effects of changes in systemic progesterone on amino acid, ion and energy substrate composition of oviduct and uterine fluids on Days 3 and 6, respectively, of the oestrus cycle in cattle. Oviduct and uterine fluids were collected in situ following infusion of progesterone. There was no effect of progesterone on oviduct fluid secretion rate; however, uterine fluid secretion rate was lowered. Progesterone increased uterine glucose, decreased oviduct sulfate and, to a lesser degree, oviduct sodium, but had no effect on any of the ions in the uterus. The most marked effect of progesterone was on oviducal amino acid concentrations, with a twofold increase in glycine, whereas in the uterus only valine was increased. These results provide novel information on the maternal environment of the early cattle embryo and provide further evidence of progesterone regulation of oviduct amino acid concentrations in cattle.

The effect of dose and route of oestradiol benzoate administration on plasma concentrations of oestradiol and FSH in long-term ovariectomised heifers

Oestradiol (E(2)) suppresses FSH and affects follicle wave dynamics in cattle. However, neither the optimum dose of ODB required to suppress FSH nor the effect of route of ODB administration on blood concentrations of E(2) are known; hence, the aim of this experiment was to answer these questions. Ovariectomised heifers received Progesterone Releasing Intravaginal Device (PRID) for 7 days, and 4 days later heifers received one of eight ODB treatments at second PRID insertion as follows; (1) 0.0 mg (Control; n=3), (2) 0.5 mg (n=4), (3) 1.0 mg (n=4), (4) 2.5 mg (n=6), (5) 5.0 mg (n=4), (6) 10. 0 mg (n=4), (7) 5.0 mg (n=4), and (8) 10.0 mg (n=5). For treatments 2-6 inclusive, ODB was administered intramuscularly in oil, while for treatments 7 and 8, the ODB in powder form was administered topically in the vagina by gelatine capsule attached to the PRID. Blood samples were collected every 6 h for the first 48 h, every 12 h for the next 48 h, and twice daily for a further 6 days. The interval from ODB administration to peak E(2) concentration was similar (P0.05) for treatments 2-6 where ODB was administered intramuscularly (mean 13.4+/-1.24 h), and was longer (P<0.05) for the intravaginal capsule treatments (mean 25.5+/-2.84 h). Plasma concentrations of E(2) increased with increasing intramuscular dose of ODB injected, (plasma E(2)=-0.237+16.109 (dose)-0.74 (dose)(2), R(2)=0.75; P<0.05). Peak plasma concentrations of E(2) following the 5- and 10-mg capsules were similar to each other and to those following the 0.5-mg injection (P0.05), but were lower than concentrations obtained following injection of 1.0-5.0 mg (P<0.05). Across all treatments, both the maximum percentage decline in FSH and the interval to FSH nadir were related to the peak plasma concentrations of E(2) (maximum % decline in FSH=11.17+1.564 (peak E(2))-0.009 (peak E(2))(2), R(2)=0.75; P<0.01), (hours to FSH nadir=10.628+1.486(hours to peak E(2))-0.0282(hours to peak E(2))(2), R(2)=0.22; P<0.05). Concentrations of FSH increased as E(2) declined from its peak value, irrespective of maximum value achieved. It was concluded that the intramuscular administration of ODB in oil to ovariectomised heifers given a PRID results in higher plasma concentrations of E(2) and causes a greater reduction in FSH than administration topically by intravaginal gelatine capsule. E(2) transiently suppresses FSH in ovariectomised heifers, and the magnitude of the suppression is dose-dependent; however FSH concentrations begin to increase 1-2 days after ODB administration while concentrations of E(2) were declining but still high.