D. O'Callaghan

EFFECT OF NUTRITION ON ENDOCRINE PARAMETERS, OVARIAN PHYSIOLOGY, AND OOCYTE AND EMBRYO DEVELOPMENT

Reproductive efficiency in high yielding dairy cows has decreased over the past 50 years, despite significant gains in genetic selection for increased milk output. One possible reason for this decline has been a change in the nutritional intake to meet the increased energy and protein demands for higher milk production. Excess energy intake in sheep will lead to significant reductions in progesterone concentrations; the effects in cattle are not so clear. Nutrition, unless radically changed, will have little effect on gonadotropin concentrations in ruminants, and this is in contrast to the situation for pigs and for primates, where very short-term nutritional changes manifest themselves in altered gonadotropin secretion. Cattle with reduced energy intake have smaller dominant follicles and more three-wave cycles, compared with animals on higher feed intakes. One of the main areas where nutrition influences reproductive efficiency is at the level of embryo production. Several studies indicate that excess energy intake reduces the response to superovulation and also decrease the yield of embryos and alters expression of some gene constructs within the developing embryo. The mechanism of this effect is not clear but indications are that the quality of the oocytes may be compromised. Indeed recent data indicate that nutritional changes around the time of mating may have detrimental effects on the establishment of pregnancy in heifers. Thus, nutritional balancing is critical for high-yielding dairy cows, in particular. The challenge remains to modify nutritional and management strategies in such cows to maintain the levels of production made possible by genetic selection and still maintain an acceptable level of fertility.

Nutritional effects on ovulation, embryo development and the establishment of pregnancy in ruminants

The effects of high and low dietary dietary intake on reproduction in female cattle and sheep will be considered at the level of the pituitary gland, ovary and uterus. In sheep, increased dietary intake for a relatively short time will increase ovulation rate, by increasing gonadotropin secretion. Dietary intake can affect steroids such as progesterone and also intra-follicular concentrations of some growth factors such as IGF-1 and IGF-2. The effects of altered energy intake on gonadotropins and steroids in cattle are not as repeatable as those in sheep but follicular growth rates can be altered. High nutrition has a negative effect on oocyte quality, with animals on ad-libitum high energy diets particularly at risk. Overfeeding can decrease embryo quality in both sheep and cattle and it appears that this results from changes primarily at the level of the follicle or oocyte. Restricted nutrition for a short time will enhance pregnancy rates in cattle; most of this benefit appears to occur if food is restricted before insemination. Thus feeding levels before mating are particularly important to subsequent reproductive success. High dietary crude protein may decrease pregnancy rate in lactating cows. In ewes and heifers supplementation with urea failed to have any effect on pregnancy rates when good quality embryos were transferred to recipient animals exposed to high dietary crude protein. In donor ewes there were adverse effects on early embryo development following urea treatment, suggesting that the mechanism affecting the reproductive process was primarily operating at the level of the oocyte. Collectively, these data identify the overall deleterious effects of high dietary intake and excess crude protein on fertility and highlight the importance of dietary intake before ovulation on the likelihood of establishing a viable pregnancy.

Effect of genetic merit, milk yield, body condition and lactation number on in vitro oocyte development in dairy cows.

The effects of milk yield, body condition score (BCS) and lactation number on the number of oocytes recovered and blastocysts formed were studied following in vitro maturation, fertilization and culture of bovine oocytes collected from 48 high and 46 medium genetic merit dairy cows in their first and third lactation. The cows were slaughtered between 125 and 229 d post partum. Ovaries were recovered, and 2- to 10-mm follicles were aspirated. Cleavage rate and number of blastocysts were determined at 44 h and 7 d after insemination, respectively. Oocytes from high genetic merit cows formed fewer blastocysts and had lower cleavage and blastocyst formation rates than those from medium genetic merit cows (0.36 +/- 0.19, 70.4 and 6.8% vs 0.85 +/- 0.22, 77.4 and 11.4%, respectively). The effect of milk production was tested by grouping cows in their third lactation into high and low groups. There was no difference in number of oocytes recovered and subsequent development into blastocysts between the cows in the high milk production group (4559 to 5114 kg, n = 20) and cows in the low yield (3162 to 3972 kg, n = 20) group (6.9 +/- 1.34 vs 8.9 +/- 1.32, respectively). The effect of BCS was tested by grouping cows in their first or third lactation into high and low groups. Cleavage and blastocyst formation rates were greater for oocytes from cows with a high BCS (3.3 to 4.0, n = 20) than a low BCS (1.5 to 2.5, n = 20) (75.7 vs 61.9% and 9.9 vs 3.0%, respectively). Cows in the first lactation yielded fewer oocytes (5.7 +/- 1.24) than cows in the third lactation (7.8 +/- 0.79). Thus, the quality of oocytes probably contributes to reduced fertility, often evident in high genetic merit dairy cows.

Effect of type and quantity of concentrates on superovulation and embryo yield in beef heifers

Embryo yield and quality can be decreased following superovulation of cattle on high levels of concentrates. Concentrate type can alter rumen fermentation patterns and so affect energy availability and thus embryo quality. The objectives of this experiment were to examine the effect of 2 levels and 2 types of concentrate on superovulatory response and embryo quality in beef heifers. Beef heifers received grass silage as a basal diet and barley at 3 kg (n = 20) or ad-libitum (n = 19), or citrus/beet pulp at 3 kg (n = 18) or ad-libitum (n = 19) as the source of concentrate supplement. Silage was available ad-libitum for heifers offered 3 kg but was restricted to 1 kg DM/day for heifers on ad-libitum concentrate intake. Both concentrates contained 14% crude protein. After 100 d, heifers were treated with an intravaginal progesterone releasing device (CIDR) for 7 d, and superovulation was initiated 60 h before CIDR withdrawal. Heifers received pFSH (a total of 265 mg NIH-FSH-P1 equivalent) administered over 8 injections at 12 h intervals with the last 2 injections administered at 12 and 24 h after CIDR withdrawal; they were inseminated at 56 and 72 h after CIDR withdrawal without reference to estrus. Heifers were slaughtered 6, 7 or 8 d after the first insemination, and embryos were flushed from the uterus with PBS and were graded morphologically. Statistical analyses were performed using analysis of variance. There was no interaction between level and type of diet, and thus data for the main effects are presented separately. Final live weights at the end of the experiment and carcass weights for heifers offered ad-libitum concentrate (643.8+/-6.9 kg; 354+/-8 kg, respectively) were higher (P<0.01) than those of heifers offered 3 kg concentrate (613.1+/-7.5 kg; 338.4+/-3.2 kg, respectively) while live weights and carcass weights of heifers offered barley (625.6+/-8.1 and 345.4+/-4.1 kg, respectively) or citrus/beet pulp concentrate (631.4+/-7.0 and 348.0+/-3.1 kg, respectively) were not different (P>0.05). Superovulatory responses (number of corpora lutea at slaughter) was greater (P<0.06) when heifers were offered 3 kg (15.5+/-1.6) than when offered ad-libitum concentrates (12.3+/-1.4). However, the superovulatory response for both citrus/beet pulp (14.4+/-1.5) and barley (13.4+/-1.5) diets were not different (P>0.05). Heifers offered 3 kg concentrates produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) compared with heifers fed ad-libitum concentrates (2.8+/-0.4). Heifers offered citrus/beet pulp produced greater (P<0.05) numbers of transferable embryos (4.8+/-0.7) than heifers offered barley (2.9+/-0.5). These data indicate that high concentrate intake has a negative effect on embryo yield and quality and that a barley compared with citrus/beet pulp based concentrate has a negative effect on embryo quality following superovulation in beef heifers.

EFFECT OF DIET QUANTITY AND UREA SUPPLEMENTATION ON OOCYTE AND EMBRYO QUALITY IN SHEEP

The objective was to investigate the effects of dietary energy and urea supplementation on oocyte and embryo quality in sheep using in vivo and in vitro experimental models. Sixty-three ewes were fed grass meal at 0.5 or 2.0 times maintenance energy requirements (MER). The diet was supplemented with feed grade urea (U) for half of the ewes on each energy treatment. Ewes were stimulated with 1000 IU eCG and either slaughtered on the day of pessary withdrawal, for in vitro embryo production, or mated and slaughtered on Day 5 for embryo recovery. Urea decreased cleavage rate (48.3 vs 39.7%) and consequently blastocyst rate (41.6 vs 36.8%) but the differences were not significant. Oocytes from animals on 2.0 MER had a lower cleavage rate (54.9 vs 36.0%) and blastocyst yield (49.3 vs 31.4%) than those on 0.5 MER. However, there was an interaction between urea and energy for cleavage (P = 0.04) and blastocyst yield (P = 0.03) indicating a variable response to urea in the presence of high energy. This was manifested by a decrease in cleavage rate in the presence of urea and high energy (22%, 8 of 36), and a reduction in blastocyst development (19%, 7 of 36). When blastocyst development rate was expressed as a proportion of cleaved oocytes there was no difference between groups; in addition, there was no difference between groups in terms of blastocyst hatching rate (overall mean 66.1%) or blastocyst cell number on Day 8 (overall mean +/- SEM, 138.4 +/- 9.0, n=61). The effect of urea on cleavage rate in vivo was more severe. Urea supplementation reduced (P<0.001) the cleavage rate (93 vs 62%). Despite this, the yield of blastocysts was unaffected. Oocytes from ewes on 0.5 MER exhibited a lower (P<0.05) cleavage rate than those on 2.0 MER (66 vs 87%). This effect was also apparent at the blastocyst stage (40.0 vs 50.9%), although the difference was no longer significant. There were no differences in hatching rate (overall mean 70.7%) or blastocyst cell numbers (overall mean +/- SEM, 166.3 +/- 15.6, n=40). Collectively, these results suggest that both high dietary energy and urea content influence subsequent embryo development in vitro, and the deleterious effects of urea are likely influenced by concomitant energy intake.

Effects of superovulated heifer diet type and quantity on relative mRNA abundances and pyruvate metabolism in recovered embryos.

This study investigated the effects of quantity and type of diet fed to superovulated donor heifers on molecular and metabolic indices of embryonic development. These effects included the relative abundances of mRNAs for the alpha 1 subunit of Na/K-ATPase and the antioxidant enzyme Cu/Zn-SOD, as well as pyruvate utilization in bovine morulae and blastocysts developed in vivo. Heifers were fed a daily ration of either grass silage and a citrus-beet pulp-based concentrate or grass silage and a barley-based concentrate for 116 days, both at 3 kg per day or ad libitum. In embryos derived from heifers fed the pulp-based diets, the relative abundances of the transcripts were not affected by either day of collection or quantity of diet. In embryos derived from heifers fed the barley-based diets, the relative abundances of the Na/K-ATPase transcripts were also not changed by these main effects, while the relative abundances of the Cu/Zn-SOD transcripts were affected by day of collection and by the quantity of diet. Pyruvate metabolism was affected by day of collection, and was significantly increased in day 8 embryos compared with day 7 and day 6 embryos. Diet quantity did not affect pyruvate utilization, whereas diet type did increase pyruvate metabolism in the barley group when compared with the pulp group. The results of this study show for the first time that molecular and metabolic variations may exist in embryos derived in vivo and developed in donor heifers on nutritional regimens differing in type and quantity. Differences in embryos collected on different developmental days may be attributed to varying cell numbers. Alterations in the relative abundances of the Cu/Zn-SOD transcripts and pyruvate metabolism caused by the quantity of diet fed to the donor animal were likely to have been due to alterations in metabolic end products that accumulate in reproductive tract fluids, whereas differences in embryonic metabolism caused by type of diet are related to the composition of the diet. These findings characterize embryos produced in vivo at the molecular level, indicating that the molecular markers used in the present study can differentiate between populations of embryos produced under different nutritional regimens and determine conditions conductive to the production of good quality embryos.

Effect of roughage type and concentrate supplementation on follicle numbers and in vitro fertilisation and development of oocytes recovered from beef heifers

Increasing dietary energy tends to decrease the ovulatory response and produce fewer viable embryos following superovulation of beef cattle. Data in sheep indicate that high energy intake can decrease progesterone concentrations (P4), although effects in cattle are not as clear. The objectives were to evaluate the effects of roughage type and concentrate supplementation on P4 concentrations, follicle growth and subsequent oocyte fertilisation and embryo development in vitro. Forty-two beef heifers were allocated to 3 treatment groups: (i) silage ad libitum plus 6 kg concentrates (silage + conc.; n = 14); (ii) silage ad libitum (silage; n = 14) or (iii) hay ad libitum (hay; n = 14) for 40 days. Oestrus was synchronised using a controlled intravaginal progesterone releasing device (CIDR) for 7 days plus prostaglandin F2 alpha (15 mg luprostiol) administered 2 days before CIDR withdrawal. Ovaries were stimulated with 600 i.u. of follicle stimulating hormone (pFSH) administered in 6 equal doses at 12-h intervals, starting 12 days after CIDR withdrawal. Daily blood samples were collected from 3 days after CIDR insertion until CIDR withdrawal, and for another 3 days prior to pFSH, for P4 determination. Oocytes were recovered postmortem 12 h after the last pFSH injection, matured, fertilised and cultured in vitro. There was no overall effect of diet (P > 0.05) on P4 concentrations. The number of follicles grown in heifers on silage + conc (18.8 +/- 3.3), silage (23.5 +/- 3.4) or hay (18.1 +/- 2.6) were not affected by the dietary treatment (P > 0.05). The percentage of oocytes fertilised from heifers on hay (88%) was higher compared to oocytes from heifers on silage (79%; P < 0.05), but was not different (P > 0.05) compared to the proportion of oocytes from heifers on silage + conc. (86%). The percentage of fertilised oocytes that cleaved was higher from heifers on silage (94%; P < 0.01) compared with oocytes from heifers on hay (82%) or silage + conc. (86%). The proportion of embryos that developed to blastocyst was not different (P > 0.05) between groups of oocytes from heifers on silage + conc. (8%), silage (14%) or hay (15%). Heifers on silage produced numerically more blastocysts (silage: 19 from 14 heifers; silage + conc.: 8 from 14 heifers; hay: 12 from 14 heifers). These results suggest that dietary treatment used prior to oocyte recovery did not significantly influence the developmental competence of the oocytes in vitro.

The effect of supplementary long days, a subcutaneous melatonin implant and exposure to a ram on the onset of the breeding season in ewes

Seasonality of breeding in sheep occurs as a consequence of exposure to photoperiodic, social and other environmental stimuli. The objective of this experiment was to determine if the onset of the breeding season in ewes could be advanced by giving supplementary long days during pregnancy, followed by a subcutaneous melatonin implant at lambing. The additional effect of exposing ewes treated in this way to a ram was also investigated. Twin bearing ewes (mean±SEM lambing date 7 March± 2 days) maintained on natural photoperiod were randomly allocated within breed to one of the following treatments: 1. (1) control, ewes maintained on natural photoperiod throughout (n=22); 2. (2) melatonin, ewes maintained on natural photoperiod throughout and given a subcutaneous melatonin implant at lambing (n=23); 3. (3) long days plus melatonin, ewes were exposed to 18 h light: 6 h dark for an average of 44 days before lambing, given a melatonin implant at lambing and then maintained on natural photoperiod (n=21); 4. (4) long days plus melatonin plus ram, as Treatment 3, followed by continuous exposure to a ram from the summer solstice (n=22). Reproductive activity was assessed from serum progesterone concentrations in blood collected once weekly from 1 June. Control ewes began their breeding season in September (median date 21 September, range 50 days). Ewes given melatonin alone became reproductively active around the same time (median date 13 September, range 77 days; P > 0.05). Ewes exposed to long days during pregnancy followed by a melatonin implant at lambing began their breeding season earlier than controls (median date 6 September, range 93 days; P<0.05). Exposure to a ram from the summer solstice in addition to treatment with long days and melatonin resulted in an additional advancement in the onset of the breeding season (median date 29 July, range 77 days) compared with ewes exposed to long days and given melatonin (P<0.01). Thus, the combined treatment of long days during pregnancy followed by a melatonin implant at lambing can advance the breeding season of ewes. Additional exposure to a ram in such ewes can further advance the onset of the breeding season.

Effect of rumen degradable bolus containing melatonin or progestagen pessary plus pregnant mare serum gonadotropin on estrus response and lambing rate in ewes

Two practical regimens designed to induce estrus and ovulation in ewes in late anestrus were compared. Forty ewes were given a soluble glass rumen bolus containing 150 mg melatonin on July 9 and were joined with two vasectomized rams on July 23 and with three fertile rams on August 6. A second group of 40 ewes was treated with an intravaginal progestagen pessary (60mg medroxy-progesterone acetate) on July 23. Following pessary removal after 12 d, ewes were given 750 IU of pregnant mare serum gonadotropin (PMSG). Five fertile rams were joined with these ewes 48 h after progestagen removal. Melatonin concentrations were determined in single blood samples collected in early afternoon of July 21. Mating dates, lambing dates and litter sizes were recorded. Date of mating was significantly later in ewes treated with melatonin compared with those treated with progestagen plus PMSG (P<0.0001). All ewes given melatonin were mated within 4 wk, and those on progestagen plus PMSG treatment within one day of fertile ram introduction. Thirty-four ewes (85%) allocated to melatonin treatment and 36 (90%) allocated to progestagen plus PMSG treatment lambed (P>0.05). Mean (+/-SEM) lambing date was later in melatonin-treated ewes (January 17+/-1.2 d) compared to those given progestagen plus PMSG (December 30+/-0.6 d; P<0.0001). Mean litter size was lower in melatonin-treated ewes (1.5+/-0.1) compared with those given progestagen plus PMSG (2.0+/-0.1; P<0.001). Plasma melatonin concentrations indicated that 9 of 40 ewes treated with melatonin had circulating melatonin concentrations of less than 16 pg/ml. It is concluded that under conditions that existed in this experiment, treatment with progestagen plus PMSG in late anestrus resulted in more synchronous mating and lambing and a higher litter size than that following administration of a soluble glass rumen-degradable bolus containing melatonin.

Breeding season and ovulation rate in ewes treated with long days in spring followed by a melatonin implant and exposure to a ram

While the onset of the breeding season in ewes can be advanced up to 7 weeks following exposure to long days (44 days) before lambing, a melatonin implant from lambing and exposure to a ram from the summer solstice, this advance is reduced by the absence of a ram. The aim of this experiment was to determine if earlier exposure to the ram and/or exposure to more long days before lambing would further advance the onset of the breeding season. A further aim was to compare the ovulation rate and duration of the breeding season between ewes with a normal and advanced onset of the breeding season due to this treatment regime. Pregnant ewes lambing in mid March were allocated to treatments as follows: (1) controls, maintained on natural photoperiod; (2) 44 LD + mel, exposed to 44 long days (18L: 6D; LD) before lambing followed by a 700-mg melatonin implant after lambing; (3) 90 LD + mel, as in treatment 2, but exposed to 90 LD before lambing; (4) 90 LD + mel + ram, as in treatment 3, including continuous exposure to a vasectomized ram from 10 April. Reproductive state was assessed from serum progesterone concentrations in blood collected at 3- to 4-day intervals from 10 April and ovulation rate was determined during the luteal phase of the first five oestrous cycles in control ewes and ewes exposed to 90 LD + mel + ram. Ewes exposed to 90 LD + mel + ram commenced the breeding season 10 weeks earlier (median date 25 July, range 28 days; P 0·05). The duration of the breeding season was longer in ewes exposed to 90 LD + mel + ram (210 (s.e. 15) days; P 0·05). Exposure of ewes to 90 LD before lambing in March, followed by a melatonin implant and exposure to the ram from 10 April advanced the onset and extended the duration of the breeding season without an overall effect on ovulation rate. The ram is an important component of this signal sequence.