Two experiments were carried out to study the effect of nutrition on embryo development in two periods in superovulated ewes (Expt 1) and on oocyte developmental capacity during the late follicular phase (Expt 2). In Expt 1, a lower superovulation response in terms of animals ovulating (P < 0.05), ovulation rate per ewe ovulating (P = 0.1) and number of good quality embryos per animal treated (P < 0.07) was noted in ewes fed an ad libitum diet compared with ewes offered control (1.5 times the daily maintenance energy requirements, 1.5 x M) or low energy (0.5 x M) diets. Nutrition also modified the morphological and functional quality of the oocytes and embryos recovered. Thus, 92% of day 4 embryos recovered from ewes offered the control diet were classified as good embryos, compared with 70 and 82% of those recovered from ewes offered the ad libitum and low diets, respectively (P < 0.05). Ewes offered the ad libitum diet had a greater percentage of poorly developed embryos compared with ewes offered the control or low diets (P < 0.05). Ewes fed the low diet tended to have more non-fertilized oocytes than ewes offered the control diet (P = 0.09). Diet of recipient ewes to which good quality embryos were transferred on day 4 did not affect embryo quality, when assessed 12 days later (day 16 of pregnancy). However, recipient diet affected prostaglandin F(2alpha) (PGF(2alpha)) production in vitro, and uterine tissue that originated from recipient ewes on the low diet secreted more PGF(2alpha) relative to uterine tissue that originated from recipients on the control diet (P < 0.05). In Expt 2, fewer total (P < 0.05) and good quality (P < 0.01) oocytes and a lower percentage of good quality oocytes (P < 0.01) were obtained from superovulated ewes offered the ad libitum diet compared with ewes offered the low diet. In addition, cleavage rate tended to be higher (51 versus 35%, P = 0.09) in ewes offered the low diet compared with ewes offered the ad libitum diet. In conclusion, changes in diet can affect the quality of the oocyte and embryo in superovulated sheep. A lower superovulation response and a decrease in the quality of oocytes and embryos indicate that ad libitum diets are highly detrimental for superovulatory programmes when compared with low and control diets. In addition, the results from the present study indicate that a low energy diet during early embryo development increased the uterine production in vitro of PGF(2alpha) which could lead to a poor uterine environment thereby compromising the development of the embryo.
JM Lozano, P Lonergan, MP Boland and D O'Callaghan
T. Sweeney, A. Donovan, J. F. Roche and D. O'Callaghan
Three experiments examined the importance of the time and duration of exposure to a long day followed by a short day photoperiod signal in initiating reproductive activity in ewes. In Expt 1, ewes were maintained on short days (8.5 h light:15.5 h dark) from 21 December interrupted with either 105 long days (18 h light:6 h dark; LD) from 9 February or 35 LD from 9 February, 16 March or 20 April. Exposure to long days followed by short days advanced the onset of reproductive activity in comparison to control ewes maintained on simulated natural photoperiod. Exposure to long days for 105 days delayed the onset of reproductive activity (August 2 ± 3 days; P < 0.05) compared with 35 days beginning on the same date (July 13 ± 5 days). The interval from the end of the long day signal to the onset of reproductive activity was shorter (P < 0.001) however, after 105 LD than after 35 LD. In Expt 2, control ewes were moved from natural photoperiod to simulated natural photoperiod on 1 November and subsequently exposed to short days from 21 December. Four other groups were also exposed to this basic photoperiodic signal sequence but it was interrupted with either 70 LD from 16 November, or 35 LD from 16 November, 21 December or 20 April. More ewes (P <0.05) initiated reproductive activity after exposure to 70 LD from 16 November and 35 LD from 21 December or 20 April compared with control ewes maintained on short days or ewes given 35 LD from 16 November. The interval from the end of long days to the onset of reproductive activity was less (P < 0.01) in ewes given 70 LD than in ewes given 35 LD. In Expt 3, ewes on natural photoperiod were given either 90 LD from 21 September, 35 LD from 21 September, 26 October, 30 November, 4 January or 8 February followed by short days. The majority of ewes that received long followed by short days after the winter solstice resumed reproductive activity. However, all photoperiod signals given between the autumn equinox and the winter solstice failed to initiate reproductive activity in ewes during the experiment. Thus we conclude that, in ewes, the reproductive neuroendocrine axis is insensitive to long days followed by short days between the autumn equinox and the winter solstice. The reproductive axis of ewes regains sensitivity to the inductive effects of long days followed by short days at a time close to the winter solstice. Between the winter and summer solstices, long days followed by short days maintain the anoestrous state and provide the cue for initiation of reproductive activity.
D O'Callaghan, H Yaakub, P Hyttel, LJ Spicer and MP Boland
The objective was to determine the effect of dietary intake on follicle and oocyte morphology in unstimulated and superovulated ewes. Fifty-four ewes were fed grass meal at 0.5, 1.0 or 2.0 times maintenance energy requirements (M) for 32 days. Oestrous cycles were synchronized using progestagen pessaries and either unstimulated or superovulated with 200 mg pig FSH. The ewes were killed and ovaries were collected either 36 or 12 h before the anticipated LH surge. Serum progesterone concentrations in ewes on day 10 after withdrawal of the pessary were lower in ewes fed 2.0M than in ewes fed 0.5M or 1.0M (P < 0.05). LH pulse frequency tended to be higher in ewes fed 2M than 1M (1.0 +/- 0.3 versus 0.3 +/- 0.2 pulses per 8 h) on day 6 after removal of the pessary but the effect was not significant. In unstimulated ewes, more follicles (>/= 3 mm) were observed when the animals were killed in ewes fed 2.0M (3.5 +/- 0.3) than in ewes fed 0.5M (2.4 +/- 0.3) or 1.0M (2.4 +/- 0.5; P < 0. 05). Fewer follicles were observed in superovulated ewes on 0.5M (7. 5 +/- 1.2) than in ewes on 1.0M (12.0 +/- 0.5) or 2.0M (12.3 +/- 1. 4; P < 0.05). Follicular fluid progesterone concentrations were higher in ewes fed 0.5M compared with those fed 1M or 2M (P < 0.05). Insulin-like growth factor (IGF)-I concentrations were higher in follicular fluid from ewes on 1M compared with either those on 0.5M or 2M (P < 0.05), whereas IGF-II concentrations were lower in follicular fluid from ewes on 2M compared with those on 1M or 0.5M (P < 0.05). Superovulation increased follicular fluid progesterone, oestradiol, IGF-I and IGF-II concentrations (P < 0.01). Concentrations of the 34, 22 and 20 kDa IGF binding proteins were lower in follicles from superovulated ewes compared with unstimulated ewes (P < 0.05). Oocytes from superovulated ewes showed abnormalities such as premature activation of cumulus expansion and vacuolation of the nucleolus and increased frequency of detachment of interchromatin-like granules from the nucleolar remnant. Collectively, these results indicate that both high and low dietary intakes can alter systemic and follicular fluid hormone concentrations. Relative to dietary effects, the effects of superovulation were greater and involved substantial increases in follicular fluid hormone concentrations and abnormal oocyte morphology.
S. D. Johnston, P. O'Callaghan, M. R. McGowan and N. J. Phillips
Collection of semen using a modified ovine artificial vagina was attempted on 90 occasions from 25 Queensland koalas (Phascolarctos cinereus adustus). Complete ejaculates consisting of a copulatory plug and sperm fraction were collected on 36 occasions (40%) from 19 animals. Seventeen of the males produced a complete ejaculate on their first or second service. Failure to collect semen (38%) and the collection of partial ejaculates (14.5%) was attributed to lack of sexual interest, aggressive behaviour towards the female by the male, the use of non-compliant teaser females or a distinct dislike of the artificial vagina. Only a few ejaculates were contaminated with urine (4.5%) or obtained after ejaculation behaviour was terminated (3%). The mean (± sem) values for the seminal characteristics of 19 complete ejaculates were: mass of copulatory plug fraction 0.78 ±0.10 g, sperm fraction volume 0.73 ± 0.10 ml, sperm concentration 165.1 ± 26.7 × 106 ml−1, pH of sperm fraction 6.7 ± 0.2, osmolarity of sperm fraction 315.0 ± 5.4 mOsm, percentage forward motility 70.7 ± 1.8%, rate of sperm movement 4.0 ±0.1 and percentage of abnormal spermatozoa 26.9 ± 2.5%. Percentages of sperm head morphotypes and tail abnormalities were documented. Although the artificial vagina technique is limited by the need for access to oestrous females, the procedure described has been shown to be a simple, reliable method of collecting semen from captive koalas.
R. M. McKeown, D. O'Callaghan, J. F. Roche and M. P. Boland
The effects of inhibin immunization on inhibin antibody titres, semen characteristics, scrotal size, fertility, FSH, LH and testosterone concentrations were determined by immunizing adult rams against bovine inhibin α1–26-Gly-Tyr conjugated to human serum albumin (n = 16) in non-ulcerative Freund's adjuvant and DEAE:dextran (1:1) or adjuvant alone (n = 16) on days 0 (29 June), 30, 60, 191, 303 and 394. Blood samples were collected and bovine inhibin α1–26-Gly-Tyr antibody titres and serum testosterone concentrations were determined. Each month, between days 174 and 417, semen was collected every 30 min to a maximum of 15 ejaculates over 7 h and scrotal circumference was measured. Ram fertility was recorded during natural service. FSH, LH and testosterone concentrations and GnRH-induced FSH and LH release were measured in a subgroup of immunized (n = 5) and control (n = 5) rams at frequent intervals. Antibody titres were variable among immunized rams (0–46% I-labelled bovine inhibin α1–26-Gly-Tyr at 1:1600 serum dilution) but mean titres were consistently higher than in control rams (P ≤ 0.001). Immunization did not alter the semen volume, output or quality of spermatozoa or ram fertility, but increased the mean scrotal circumference (37.6 ±0.8 cm versus 34.4 ± 0.7 cm, P < 0.001). Mean FSH concentrations were higher in immunized rams during two intensive blood sampling periods (in June and August) (5.8 ± 0.7 ng ml−1 versus 3.0 ± 0.3 ng ml−1, P < 0.001 in June; and 4.8 ± 0.9 ng ml−1 versus 2.0 ± 0.3 ng ml−1, P < 0.02 in August), and were correlated with antibody titres (r 2 = 0.3, P < 0.05 in June; and r 2 = 0.8, P < 0.001 in August). Discrete FSH pulses were not detected. Immunization did not alter mean or basal testosterone or LH concentrations, or LH pulse frequency; LH pulse amplitude was increased (1.6 ± 0.2 ng ml−1 versus 0.8 ± 0.2 ng ml−1, P < 0.02) and was correlated with antibody titres (r 2=0.6, P < 0.01). Immunization enhanced GnRH-induced FSH (P <0.05) but not LH release. In conclusion, immunization of adult rams against bovine inhibin α1–26 Gly-Tyr increased scrotal circumference, mean FSH concentrations and LH pulse amplitude, without altering semen characteristics, fertility, mean LH concentrations, LH pulse frequency or mean testosterone concentrations.
D. O'Callaghan, F. J. Karsch, M. P. Boland, J. P. Hanrahan and J. F. Roche
Summary. Photoperiod may regulate seasonal reproduction either by providing the primary driving force for the reproductive transitions or by synchronizing an endogenous reproductive rhythm. This study evaluated whether breed differences in timing of the reproductive seasons of Finnish Landrace (Finn) and Galway ewes are due to differences in photoperiodic drive of the reproductive transitions or to differences in photoperiodic synchronization of the endogenous rhythm of reproductive activity. The importance of decreasing photoperiod after the summer solstice in determining the onset and duration of the breeding season was tested by housing ewes from the summer solstice in either a simulated natural photoperiod or a fixed summer-solstice photoperiod (18 h light:6 h dark; summer-solstice hold). Onset of the breeding season within each breed did not differ between these photoperiodic treatments, but Galway ewes began and ended their breeding season earlier than Finn ewes. The duration of the breeding season was shorter in Galway ewes on summer-solstice hold than on simulated natural photoperiod; duration did not differ between photoperiodic treatments in Finn ewes. The requirement for increasing photoperiod after the winter solstice for initiation of anoestrus was tested by exposing ewes from the winter solstice to either a simulated natural photoperiod or a winter-solstice hold photoperiod (8·5 h light:15·5 h dark). Onset of anoestrus within each breed did not differ between these photoperiodic treatments, but the time of this transition differed between breeds. These observations suggest that genetic differences in timing of the breeding season in Galway and Finn ewes do not reflect differences in the extent to which photoperiod drives the reproductive transitions, because neither breed requires shortening days to enter the breeding season or lengthening days to end it at appropriate times. These findings are consistent with the hypothesis that photoperiod synchronizes an endogenous rhythm of reproductive activity in both breeds and that genetic differences in timing of the breeding season reflect differences in photoperiodic synchronization of this rhythm.
Keywords: breeding season; sheep; photoperiod
D. O'Callaghan, A. Donovan, S. J. Sunderland, M. P. Boland and J. F. Roche
Four experiments were carried out to determine the effect of the presence of ewes and rams on the reproductive state of ewes. In Expt 1, the breeding season of ewes kept with a vasectomized ram ended later (April 18 ± 8 days; mean ± sem) than that of ewes isolated from rams (6 March ± 7 days; P < 0.01). In Expt 2, the end of the breeding season was later (5 May ± 6 days; P < 0.05) and the onset of the next breeding season earlier (29 September ± 2 days; P < 0.001) in ewes maintained with rams, compared with ewes isolated from rams (14 April ± 7 days and 1 November ± 2 days, respectively). There was no difference in the timing of, or variation in, reproductive transitions between ewes maintained either as individuals or in groups. In Expt 3, all ewes exposed to artificial short days from the date of the winter solstice and interrupted with 35 long days in spring resumed cyclicity (median date, 7 September; range, 59 days). Most ewes (seven of nine) exposed to short days from the date of the winter solstice and isolated from other ewes did not resume cyclicity in the following 11 months. In contrast, all ewes resumed cyclicity (median date, 19 October; range, 144 days) when exposed to short days but housed in social contact with other ewes that became reproductively active in early September; however, the onset of cyclicity was later than in ewes exposed to long days (P < 0.01). In Expt 4, the number of LH pulses per 6 h in ewes exposed to rams was higher (P < 0.001) and the time of first ovulation earlier (16 August ± 5 days; P < 0.05) than it was in ewes that were isolated from rams and exposed to either oestrous or anoestrous ewes. We conclude that there was a chronic stimulus from rams to ewes that increased the duration of the breeding season and decreased anoestrus. There was no acute effect of introduction of oestrous ewes to anoestrous ewes on LH pulse frequency and time of first ovulation of the breeding season under the natural photoperiod, and the onset of the breeding season of housed anoestrous ewes exposed to a constant photoperiod was advanced by housing them with cyclic ewes. These results highlight a role for social or other animal-related stimuli in seasonal reproduction in ewes.
C Wrenzycki, P De Sousa, EW Overstrom, RT Duby, D Herrmann, AJ Watson, H Niemann, D O'Callaghan and MP Boland
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.
S D Johnston, P O'Callaghan, K Nilsson, G Tzipori and J D Curlewis
The koala ovulates in response to mating. The purpose of this study was to document the LH surge induced by copulation and to investigate the potential roles of mechanical stimulation of the urogenital sinus and deposition of semen in induction of the luteal phase. In experiment 1, serial blood samples from four koalas that underwent normal mating showed elevated concentrations of LH approximately 24–32 h post-coitus. There was no corresponding elevation in LH in koalas (n = 4) that were exposed to the presence of a male but received no physical contact. In experiment 2, koalas on day 2 of oestrus were exposed to one of the following treatments (n = 9 per group): artificial insemination with 1 ml 0.9% sterile saline (control group), insemination with 1 ml koala semen, stimulation of the urogenital sinus with a purposebuilt glass rod (designed to mimic the action of the penis during natural mating) and urogenital stimulation with the glass rod followed by insemination of 1 ml koala semen. Confirmation of a luteal phase was based on evidence of a prolonged return to oestrus, parturition and/or elevated progesterone concentrations. Insemination of saline (0/9) and urogenital stimulation (0/9) failed to induce a luteal phase. Insemination of semen without glass rod stimulation resulted in a luteal phase in 4/9 koalas, three of which gave birth. Insemination of semen in combination with urogenital stimulation produced a luteal phase in 7/9 koalas, four of which gave birth. Semen had a significant effect on induction of the koala luteal phase (P < 0.001) but glass rod stimulation had no such effect (P = 0.335). It was concluded that semen must be involved in the induction of a luteal phase in the koala. The results presented in this study will serve to improve optimal timing and induction of ovulation for artificial insemination in the koala.
A. R. Scanlon, S. J. Sunderland, T. L. Martin, D. Goulding, D. O'Callaghan, D. H. Williams, D. R. Headon, M. P. Boland, J. J. Ireland and J. F. Roche
Two experiments were conducted in cyclic beef heifers to determine whether active immunization against bovine inhibin α 1–26 Gly-Tyr (bINH) affected follicular dynamics, hormone concentration or ovulation rate. In Expt 1, heifers (n = 9) were actively immunized against bINH conjugated to human α globulins (HAG) using bisdiazotized benzidine in non-ulcerative Freund's adjuvant (NUFA; primary on day 0; booster injections on days 53, 84 and 116 using conjugated bINH and on days 176 and 366 using unconjugated bINH; ten heifers were used as controls). Ovaries were examined daily using ultrasound scanning (days 70–155 and 384–391) and corresponding blood samples were collected for bINH antibody titre, luteinizing hormone (LH), follicle-stimulating hormone (FSH) and oestradiol determinations. Four treated and four control heifers were injected with 10 μg gonadotrophin-releasing hormone (GnRH) on day 386 (day 2 of the oestrous cycle). Although bINH-immunized heifers had variable antibody titres ranging from 4 to 50% I125-labelled bINH bound to serum diluted 1:2000, ovulation rate was unaffected. In oestrous cycles with three dominant follicles, the ovulatory follicles grew faster (2.5 ± 0.2 versus 1.6 ± 0.3 mm day−1; mean ± sem), had shorter durations of growth (5.7 ± 0.8 versus 9.6 ± 1.6 days) and duration of detection (7.5 ± 0.8 versus 12.0 ± 2.4 days) in immunized heifers. Mean concentrations of FSH, LH and oestradiol were unaltered in most cases during oestrous cycles in bINH-immunized compared with control heifers. There was no significant difference in the percentage increase in FSH or LH, after GnRH injection, between control and immunized heifers. As ovulation rate was unaltered in the first experiment, a second similar study was designed using a different immunization protocol. In Expt 2, heifers were immunized with bINH conjugated to human serum albumin using glutaraldehyde with the following doses: 0.0 (control; n = 7), 0.33 (n = 7), 1.0 (n = 8) and 3.0 (n = 7) mg. Three booster immunizations were given 33, 66 and 209 days after primary immunization. Immunization increased the number of oestrous cycles with multiple ovulations (42 of 132 (32%) oestrous cycles examined) compared with controls (1 of 30 (3.3%) oestrous cycles examined). Neither titre nor ovulation rate was affected by dose of bINH used. In summary, following bINH immunization, ovulation rate was not increased despite changes in follicular dynamics in Expt 1, but was increased in 32% of oestrous cycles in Expt 2. We conclude that immunization protocols can affect responsiveness of heifers to bINH immunization, and that immunization against inhibin can increase ovulation rate.