Effects of short and long - term nutrition and progesterone supplementation on the success of fixed - time artificial insemination in the ewe.

The success of fixed - time artificial insemination (AI) in the ewe is variable due to poor synchrony of estrus. We examined the effects of long-term nutrition (LTN; low, medium, high - 6 months), short-term nutrition (STN; 1.0 M, 1.5 M - 14 days) and progesterone supplementation (P; single pessary, replacement on Day 9) on synchrony and reproductive outcomes. High LTN advanced (P < 0.05) estrus, increased (P = 0.06) pregnancy (range 71.1 - 81.1%) and improved (P < 0.01) litter size (range 1.30 - 1.50). STN increased (P < 0.05) pregnancy (79.0 versus 72.3%) but not litter size or timing of estrus. A LTN x STN interaction (P < 0.01) for time of estrus indicates that the effects of LTN were moderated by STN depending on the level of LTN. Pessary replacement delayed (P < 0.05) the onset of estrus, improved synchrony but did not affect pregnancy or litter size. High LTN increased (P < 0.05) the number of large (≥ 3.8 mm) and medium - size follicles (2.0 - 3.7 mm) but the diameter of large follicles tended to be reduced (P = 0.08) on Day 12. STN did not affect follicle number or size whilst P reduced (P < 0.05) the diameter of large follicles on Day 12 (4.83 versus 5.10 mm) and increased the number of medium - size follicles (3.56 versus 2.74 mm). In conclusion, both LTN and STN are major sources of variability in AI programs whilst pessary replacement has potential to reduce variability.

Heat stress from current and predicted increases in temperature impairs lambing rates and birth weights in the Australian sheep flock.

Livestock heat stress threatens production, particularly in semi-arid, arid and tropical regions. Using established temperature thresholds for sheep, we modelled +1 °C and +3 °C temperature increases over the historical baseline, estimating that 2.1 million potential lambs are lost annually due to heat stress alone, increasing to 2.5 and 3.3 million, respectively, as temperatures rise. Heat stress poses risks at key periods of the reproductive cycle, with consequences across the Australian sheep flock.

Strategies to improve the success of fixed-time artificial insemination in the ewe.

During the sheep breeding season, ovulatory follicles vary widely in age at pessary removal impacting both the timing of oestrus and pregnancy rates following artificial insemination (AI). Ovulatory follicles that emerge between days 7 to 9 of the pessary period are associated with higher fertility whilst those that emerge earlier or later are associated with lower fertility. In this study, two strategies to improve the success of AI by controlling the development of the ovulatory follicle were examined. In the first, ewes were treated with PGF2α at either -12 and/or +6 days (experiment 1) or -27 days (experiment 2) relative to pessary insertion to control the time of emergence of the ovulatory follicle. In the second, ewes were treated with eCG (400 IU per ewe) at either 0 h, -6 h or -12 h relative to pessary removal (experiment 3) to improve the development of young ovulatory follicles. PGF2α administered on day -27 increased the percentage of pregnant ewes by 17.8% and the number of foetuses per 100 ewes inseminated by 33.9%. PGF2α treatment at other times had either no effect or reduced fertility. During the breeding season, treatment with eCG at -12 h improved the synchrony of oestrus, reduced the size of the ovulatory follicle but did not improve pregnancy rate compared with other treatments. Treatment had no effect during the non-breeding season, supporting earlier findings that the quality of young ovulatory follicles differs during the year. In conclusion, PGF2α treatment 27 days before pessary insertion provides a new and cheap strategy to improve the success of fixed-time AI programs.

Ovarian follicle dynamics in ewes treated with intra-vaginal progesterone pessaries. 2. Factors affecting timing of estrus and reproductive outcomes following artificial insemination.

This study extends observations on the effects of intra-vaginal progesterone treatment on the relationships between the time of luteolysis, emergence of the ovulatory follicle, timing of estrus and ewe fertility. Observations were made in progesterone - treated ewes in autumn, the spring equinox and late spring (Experiment 1, Data set 1) and in progesterone - treated ewes and naturally cycling ewes in autumn and the spring equinox (Experiment 1, Data set 2). In Data set 1, the day of emergence of both the first and second ovulatory follicle was positively related to the day luteal regression within each season. In turn, the day of emergence influenced the timing of estrus by means of a season by day of luteal regression interaction (P < 0.001) indicating that the relationship was positive in autumn and the spring equinox but negative in late spring. In autumn, older ovulatory follicles were associated with an earlier onset of estrus compared with younger ovulatory follicles. In late spring, this relationship was reversed and was influenced by whether or not ewes were cycling at the time of pessary insertion. In Data set 2, the relationship between the day of follicle emergence and luteal regression was influenced by a treatment by day of regression interaction indicating the relationship was positive in treated ewes and negative in naturally cycling ewes. Timing of estrus was positively related (P < 0.001) to both the day of luteal regression and the day of follicle emergence (P < 0.05), with both relationships being stronger in naturally cycling ewes than in treated ewes. In Experiment 2, pregnancy rate following artificial insemination in autumn was highest (90.2%) when luteolysis occurred during Days 7-9 of the pessary period compared with Days 1-6 (77.8%, P = 0.16), 10 to 12 (68.8%, P < 0.05) or Days ≥13 (71.2%, P < 0.05). Timing of estrus was not affected. The mean diameter of ovulatory follicles that emerged during Days 7-9 was larger on Day 12 (5.8 ± 0.13 mm) compared with other periods (range 4.7 ± 0.05 to 5.6 ± 0.14 mm). This study provides two potential strategies to improve the success of AI programs. Firstly, appropriately timed treatment with PGF2α to control the time of emergence of ovulatory follicles and, secondly, earlier treatment with eCG to improve the development of ovulatory follicles that emerge late in the pessary period. Each is likely to be influenced by season and the cyclical status of the ewe.

Ovarian follicle dynamics in ewes treated with intra-vaginal progesterone pessaries. 1. Follicle waves and parameters of the estrous cycle.

Progesterone treatment for synchrony of estrus is standard in sheep artificial insemination (AI) programs but can be associated with poor outcomes. Potential for improvement exists through a better understanding of the interactions between follicle development, luteal regression, emergence of the ovulatory follicle and timing of estrus. These interactions were examined by comparing progesterone-treated (Day 1 = day of pessary insertion) and naturally cycling ewes (Day 1 = day after estrus) at three times of the year (Autumn, Spring equinox and late Spring). Observations were made from Day 1 until the day of ovulation. Compared with the natural cycle, progesterone treatment (300 mg intra-vaginal pessary for 14 d) reduced the number of follicle waves (2.2 ± 0.18 versus 2.8 ± 0.12; P < 0.05) and increased the length of the ovulatory wave (8.6 ± 0.45 versus 6.6 ± 0.42 d; P < 0.05). The number of follicles per wave, the inter-wave interval and ovulation rate were not affected. However, progesterone treatment induced (P < 0.05) an earlier luteolysis (9.7 ± 0.51 versus 15.4 ± 0.49 d after Day 1), an earlier emergence of the ovulatory follicle (7.5 ± 0.48 versus 11.4 ± 0.46 d after Day 1) and an earlier onset of estrus (26.1 ± 2.95 versus 53.3 ± 2.84 h after Day 14). Time of year also influenced the response to progesterone treatment. In Autumn compared with the Spring equinox and late Spring, there was a reduction (P < 0.05) in follicle wave number (2.4 ± 0.21 versus 2.5 ± 0.29 versus 3.0 ± 0.20 respectively), follicles per wave (2.6 ± 0.27 versus 3.5 ± 0.25 versus 3.2 ± 0.20 respectively), ovulation rate (1.6 ± 0.12 versus 1.9 ± 0.12 versus 2.0 ± 0.10 respectively) and the inter-wave interval was longer (5.3 ± 0.40 versus 4.0 ± 0.32 versus 3.8 ± 0.27 d respectively; P < 0.05). Time of year also influenced (P < 0.05) the time of luteolysis (earliest in late Spring), emergence of the ovulatory follicle (earliest in Autumn) and onset of estrus (earliest in Autumn). It is concluded that (1) the effects of progesterone treatment on follicle waves are relatively minor, (2) the effects of treatment on timing of luteolysis, emergence of the ovulatory follicle and onset of estrus are all significant although the effects on AI outcomes remain to be determined and (3) time of year has a minimal effect on follicle waves but a more significant effect on other parameters of the estrous cycle. A better understanding of these complexities will assist in the development of improved protocols for synchrony of estrus.

Review of the impact of heat stress on reproductive performance of sheep.

Heat stress significantly impairs reproduction of sheep, and under current climatic conditions is a significant risk to the efficiency of the meat and wool production, with the impact increasing as global temperatures rise. Evidence from field studies and studies conducted using environmental chambers demonstrate the effects of hot temperatures (≥ 32 °C) on components of ewe fertility (oestrus, fertilisation, embryo survival and lambing) are most destructive when experienced from 5 d before until 5 d after oestrus. Temperature controlled studies also demonstrate that ram fertility, as measured by rates of fertilisation and embryo survival, is reduced when mating occurs during the period 14 to 50 d post-heating. However, the contribution of the ram to heat induced reductions in flock fertility is difficult to determine accurately. Based primarily on temperature controlled studies, it is clear that sustained exposure to high temperatures (≥ 32 °C) during pregnancy reduces lamb birthweight and will, therefore, decrease lamb survival under field conditions. It is concluded that both ewe and ram reproduction is affected by relatively modest levels of heat stress (≥ 32 °C) and this is a concern given that a significant proportion of the global sheep population experiences heat stress of this magnitude around mating and during pregnancy. Despite this, strategies to limit the impacts of the climate on the homeothermy, behaviour, resource use and reproduction of extensively grazed sheep are limited, and there is an urgency to improve knowledge and to develop husbandry practices to limit these impacts.

Caffeine: A potential strategy to improve survival of neonatal pigs and sheep.

Caffeine is commonly used to treat pre-and postnatal injuries, including apnoea in premature infants, as well as neurological impairment caused by hypoxia or asphyxiation often associated with difficult birthing. As an adenosine antagonist, caffeine is metabolised rapidly and transported into many tissues. Caffeine stimulates the brain respiratory centre, improving respiratory function in immature infants or neonates, provides neuroprotection to the fetal brain, and initiates non-shivering thermoregulation increasing metabolic rates. Recently, potential benefits of caffeine for animal production have been investigated. This has particularly occurred in pig production, where large litters are associated with relatively long parturition durations, and piglets born near the end of the parturition period have an increased risk of mortality due to asphyxia-related birthing injury. Similarly, in sheep, dystocia or prolonged parturition is a significant problem, where neonatal injury, dystocia and death in utero contributes to approximately 46 % of lamb mortalities. Within these two livestock production systems, large prevalence's of neonatal mortality is a persistent issue contributing to lost revenue, as well as being a significant animal welfare concern. Pre-partum maternal caffeine supplementation is a promising strategy to reduce neonatal mortality; however, there needs to be refinement of appropriate quantities administered, duration and administration pathway to provide producers with an efficient and cost-effective method to reduce mortality rates and increase production output. The information in this review details effects, benefits and important considerations regarding caffeine use in animal production, and identifies areas of limited knowledge where further research is needed.

Impact of in vitro embryo culture and transfer on blood pressure regulation in the adolescent lamb.

Nutrition during the periconceptional period influences postnatal cardiovascular health. We determined whether in vitro embryo culture and transfer, which are manipulations of the nutritional environment during the periconceptional period, dysregulate postnatal blood pressure and blood pressure regulatory mechanisms. Embryos were either transferred to an intermediate recipient ewe (ET) or cultured in vitro in the absence (IVC) or presence of human serum (IVCHS) and a methyl donor (IVCHS+M) for 6 days. Basal blood pressure was recorded at 19-20 weeks after birth. Mean arterial pressure (MAP) and heart rate (HR) were measured before and after varying doses of phenylephrine (PE). mRNA expression of signaling molecules involved in blood pressure regulation was measured in the renal artery. Basal MAP did not differ between groups. Baroreflex sensitivity, set point, and upper plateau were also maintained in all groups after PE stimulation. Adrenergic receptors alpha-1A (αAR1A), alpha-1B (αAR1B), and angiotensin II receptor type 1 (AT1R) mRNA expression were not different from controls in the renal artery. These results suggest there is no programmed effect of ET or IVC on basal blood pressure or the baroreflex control mechanisms in adolescence, but future studies are required to determine the impact of ET and IVC on these mechanisms later in the life course when developmental programming effects may be unmasked by age.

Neonatal lamb mortality: major risk factors and the potential ameliorative role of melatonin.

High incidences of pre-weaning mortality continue to limit global sheep production, constituting a major economic and welfare concern. Despite significant advances in genetics, nutrition, and management, the proportion of lamb deaths has remained stable at 15-20% over the past four decades. There is mounting evidence that melatonin can improve outcomes in compromised ovine pregnancies via enhanced uterine bloodflow and neonatal neuroprotection. This review provides an overview of the major risk factors and underlying mechanisms involved in perinatal lamb mortality and discusses the potential of melatonin treatment as a remedial strategy. Supplementing pregnant ewes with melatonin enhances uterine bloodflow and fetal oxygenation, and potentially birthweight and neonatal thermogenic capacity. Melatonin freely crosses the ovine placenta and blood-brain barrier and provides neuroprotection to the fetal lamb during periods of chronic and acute hypoxia throughout gestation, with improved behavioural outcomes in hypoxic neonates. The current literature provides strong evidence that maternal melatonin treatment improves outcomes for lambs which experience compromised in utero development or prolonged parturition, though to date this has not been investigated in livestock production systems. As such there is a clear basis for continued research into the effects of maternal melatonin supplementation during gestation on pre-weaning survival under extensive production conditions.

Supplementing Merino ewes with melatonin during the last half of pregnancy improves tolerance of prolonged parturition and survival of second-born twin lambs.

High preweaning mortality rates continue to limit sheep production globally, constituting a major economic and welfare concern. Greater losses in twin lambs (≥30%) compared with singletons (≥10%) are attributed primarily to lower birth weight and increased risk of intrapartum hypoxia, leading to impairment of thermoregulation, neuromotor activity, and maternal bonding behavior. Previous intensive studies demonstrated that supplementing pregnant ewes with melatonin reduced the adverse effects of fetal growth restriction and perinatal hypoxia on the neonatal brain via increased umbilical blood flow, placental efficiency, and antioxidant actions. The current study examined the effects of supplementing pregnant ewes with melatonin on lamb survival, birth weight, and behavior under intensive conditions. From gestational day (gD) 80 until parturition, pregnant singleton and twin-bearing ewes were supplemented with melatonin via a 2-mg capsule fed daily (Mel-FED, n = 61) or 18 mg subcutaneous implant (Regulin), with one implant administered at gD80 and another at gD125 (Mel-IMP, n = 60). Control ewes received no supplementation (CTL, n = 60). Ewes and lambs were monitored via video throughout parturition. Postpartum measures were taken from lambs at 4 and 24 h (live weight [LW], rectal temperature, serum immunoglobulin G, and latency to stand and suck after birth) and LW at 72 h, 7 d, marking (49.7 ± 0.2 d), and weaning (124.2 ± 0.8 d). Chi-square analysis was used to compare lamb survival between treatment groups. There were no treatment effects on singleton lamb survival. Melatonin supplementation tended to increase the proportion of twin lambs surviving from birth to weaning (Mel-FED = 85.5%; Mel-IMP = 85.9%; CTL = 72.9%; each P < 0.1). Survival of first-born twins did not differ between treatment (each ~90%, P = 0.745) but within second-born twins, survival of Mel-FED was greater than CTL (81.6 vs. 57.1%, P = 0.023), and Mel-IMP (78.1%) tended to be greater than CTL (P = 0.068). Similarly, in second-born twins exposed to prolonged parturition (≥ 90 min), survival of lambs from Mel-FED ewes was greater than CTL (86.7% vs. 42.9%, P = 0.032), while Mel-IMP was intermediate (66.7%). These data suggest that the neuroprotective actions of melatonin may improve twin lamb survival by increasing tolerance of prolonged parturition and provide a sound basis for continued testing in extensively managed sheep flocks.

Maternal melatonin implants improve twin Merino lamb survival.

High preweaning mortality rates cost the Australian sheep industry an estimated $540 million annually in lost production, with losses significantly greater in twin (≥30%) compared with singleton lambs (≥10%). Previous intensive studies demonstrated that supplementing pregnant ewes with melatonin reduces adverse effects of fetal growth restriction and perinatal hypoxia on the neonatal brain via increased umbilical blood flow, placental efficiency, and antioxidant actions. The current study examined the effects of supplementing ewes with melatonin on the survival of twin Merino lambs under extensive grazing conditions. Pregnant mixed age ewes were implanted with 1 (M1, n = 50) or 2 (M2, n = 53) slow-release melatonin implants (18 mg, Regulin) at gestational days 70 to 90. Control ewes received no supplementation (CTL, n = 54). Ewes were monitored twice daily throughout the lambing period. Lamb survival, weight, and rectal temperature were recorded on the day of birth. Lamb blood samples were taken the following day for serum immunoglobulin G (IgG) analysis. Lamb survival and weight were recorded again at marking (30.6 ± 0.6 d postpartum) and weaning (70.7 ± 0.6 d postpartum). Lamb survival was increased in both melatonin treatments to 3 d postpartum (M1 = 98.0%; M2 = 95.3%; CTL = 83.3%; each P < 0.01), and this improvement was maintained to weaning (M1 = 94.0%; M2 = 92.5%; CTL = 79.6%; each P < 0.01). Melatonin did not affect lamb birthweight, rectal temperature, or growth rate. However, the rates of parturition-related death (dystocia, stillbirth, and birth injury) were greater in CTL lambs than M1 (P = 0.009) and M2 (P = 0.035). This suggests that improved survival is primarily due to melatonin-induced neuroprotection, although further studies are required to clarify the underlying mechanisms. These data provide evidence that supplementing pregnant twin-bearing Merino ewes with melatonin may be a practical strategy to reduce neonatal mortality and improve weaning rates in extensively managed sheep flocks. Although the present data are promising, this study is limited by small sample size and requires further replication.

Maternal Supplementation with Dietary Betaine during Gestation to Improve Twin Lamb Survival.

Betaine increases the synthesis of creatine, an energy-rich amino acid that increases adenosine triphosphate (ATP) and has neuroprotective properties which may improve post-natal lamb survival. This study determined whether maternal betaine supplementation during gestation would improve body weight, thermoregulation, time to stand and suck, colostrum intake and survival to weaning of twin lambs. Twin-bearing Merino ewes received dietary betaine at either 0 g/day (Control, CTL), 2 g/day from ram introduction to parturition (Early betaine, EB) or 4 g/day from Day 80 of gestation to parturition (Late betaine, LB). Ewes were housed individually during parturition and measures were collected at 4, 24 and 72 h and Day 7 post-partum, and at marking (53.2 ± 0.2 days of age) and weaning (99.3 ± 0.2 days of age). The EB treatment resulted in heavier lambs at weaning compared with CTL and LB lambs (p < 0.05). Time to stand and suck from birth was longer in EB lambs (p < 0.05), whereas, the interval from birth to first suck was shorter for LB lambs (p < 0.05). Lamb survival rate was the highest for LB lambs at 72 h and Day 7 (p < 0.05), and lowest for EB lambs on Day 7 (p < 0.05). These data indicated that betaine supplementation at 4 g/day during the second half of pregnancy improved twin lamb survival to Day 7 and shortened the interval from birth to first suck; whereas feeding ewes 2 g/day of betaine for the duration of pregnancy increased twin lamb body weight at weaning, but increased both the time to attain behavioural milestones and mortalities before Day 7.

Plasma anti-Müllerian hormone concentration as a predictive endocrine marker for selection of donor lambs to improve success in juvenile in vitro embryo transfer programs.

The use of juvenile invitro embryo transfer (JIVET) is limited by variation between prepubertal lambs in ovarian response to exogenous gonadotrophins. In cattle, anti-Müllerian hormone (AMH) is a predictive endocrine marker of antral follicle count. In this study we measured plasma AMH concentrations in lambs at 3 and 5 weeks of age and determined associations between AMH concentrations and ovarian response to gonadotrophins and invitro blastocyst production at 6-8 weeks of age in a JIVET program. At 5 weeks, AMH (n=38) was positively correlated with surface antral follicle count (r=0.87, P<0.001), blastocysts produced (r=0.92, P<0.001) and blastocysts produced as a proportion of oocytes collected (r=0.44, P<0.01) or cleaved (r=0.43, P<0.01). Similar associations were observed between AMH at 3 weeks (n=30) and follicle number (r=0.70, P<0.05) and blastocysts produced (r=0.87, P<0.05). Lambs with high (>2.2ngmL-1) compared with medium (0.4-2.2ngmL-1) and low (<0.4ngmL-1) AMH at 5 weeks had more antral follicles (mean (±s.e.m.) 118.7±13.9 vs 68.2±8.1 and 30.4±12.3 respectively; P<0.05) and more blastocysts produced (mean (±s.e.m.) 54.9±6.9 vs 18.9±4.0 and 7.5±6.1 respectively; P<0.05). These results suggest that AMH concentration at 5 weeks of age can be used to select donor lambs which enhance the success of JIVET programs.

Simulated shift work disrupts maternal circadian rhythms and metabolism, and increases gestation length in sheep.

KEY POINTS: Shift work impairs metabolic health, although its effects during pregnancy are not well understood We evaluated the effects of a simulated shift work protocol for one-third, two-thirds or all of pregnancy on maternal and pregnancy outcomes in sheep. Simulated shift work changed the timing of activity, disrupted hormonal and cellular rhythms, and impaired maternal glucose tolerance during early pregnancy. Gestation length was increased in twin pregnancies, whereas singleton lambs were lighter at a given gestational age if mothers were subjected to shift work conditions in the first one-third of pregnancy. Exposure to rotating night and day shifts, even if only in early pregnancy, may adversely affect maternal metabolic and pregnancy outcomes. ABSTRACT: Shift workers are at increased risk of developing type 2 diabetes and obesity; however, the impact during pregnancy on maternal metabolism is unknown. Using a large animal model, we assessed the impact of simulated shift work (SSW) exposure during pregnancy on maternal circadian rhythms, glucose tolerance and pregnancy outcomes. Following mating, ewes were randomly allocated to a control photoperiod (CON 12 h light, 12 h dark) or to SSW, where the timing of light exposure and food presentation was reversed twice each week for one-third, two-thirds or all of pregnancy. Maternal behaviour followed SSW cycles with increased activity during light exposure and feeding. Melatonin rhythms resynchronized within 2 days of the photoperiod shift, whereas peripheral circadian rhythms were arrhythmic. SSW impaired glucose tolerance (+29%, P = 0.019) and increased glucose-stimulated insulin secretion (+32%, P = 0.018) in ewes with a singleton fetus in early but not late gestation. SSW exposure did not alter rates of miscarriage or stillbirth, although it extended gestation length in twin pregnancies (+2.4 days, P = 0.032). Relative to gestational age, birth weight was lower in singleton progeny of SSW than CON ewes (-476 g, P = 0.016). These results have implications for the large number of women currently engaged in shift work, and further studies are required to determine progeny health impacts.

Phyto-oestrogens affect fertilisation and embryo development in vitro in sheep.

Phyto-oestrogens such as isoflavones are natural compounds that can profoundly affect reproductive function. In the present study, we tested whether including isoflavone compounds (genistein, biochanin A, formononetin) in the maturation medium would affect the outcomes for ovine oocytes in vitro. Each isoflavone compound was evaluated at five concentrations (0, 2.5, 5, 10, 25µgmL-1) and the entire protocol was repeated four times. Cumulus-oocyte complexes were randomly allocated to the treatments, then fertilised and cultured in vitro. Compared with control (0µgmL-1), the lower concentrations of isoflavone (2.5, 5 and 10µgmL-1) had no detectable effect on the rates of cleavage or embryo development, or on embryo total cell counts (TCC). However, the highest concentration (25µgmL-1) of all three isoflavones exerted a variety of effects (P<0.05): genistein decreased cleavage rate, blastocyst rate and blastocyst efficiency (blastocysts produced per 100 oocytes); biochanin A decreased cleavage rate and blastocyst efficiency; and formononetin decreased blastocyst rate and blastocyst efficiency. Biochanin A (25µgmL-1) reduced embryo TCC specifically at the hatched blastocyst stage (P<0.05). We conclude that the presence of isoflavones at 25µgmL-1 during IVM decreases the cleavage rate and inhibits blastocyst hatching.

Sex of co-twin affects the in vitro developmental competence of oocytes derived from 6- to 8-week-old lambs.

Several intrinsic factors (age, genotype, liveweight) influence the reliability of juvenile in vitro fertilisation embryo transfer (JIVET) programs. Limited evidence indicates that variability between lambs is reduced in twin-born lambs. We examined the impact of birth type (single, twin, triplet) and sex of the co-twin (with age, birthweight and liveweight as covariates) on JIVET outcomes. Birth type did not influence any parameter studied. However, blastocysts produced, as a percentage of embryos cleaved or total cumulus-oocyte complexes collected, was higher (P<0.05) for females born with a female co-twin (67.0±6.1, 57.5±6.0 respectively) compared with those born with a male co-twin (26.9±6.5, 22.3±6.2 respectively; least-square mean±s.e.m.). Blastocyst rates for lambs born with a male co-twin did not differ significantly from lambs born either as singles (39.5±6.7%, 34.6±6.5% respectively) or triplets (43.1±10.6%, 36.5±10.3% respectively). Other parameters were not influenced by sex of the co-twin. These results are indicative of an enhancement effect of the female co-twin on oocyte development. From a practical perspective, selecting lambs for a JIVET program based on litter size and sex of the co-twin is warranted.

Impact of maternal undernutrition around the time of conception on factors regulating hepatic lipid metabolism and microRNAs in singleton and twin fetuses.

We have investigated the effects of embryo number and maternal undernutrition imposed either around the time of conception or before implantation on hepatic lipid metabolism in the sheep fetus. We have demonstrated that periconceptional undernutrition and preimplantation undernutrition each resulted in decreased hepatic fatty acid ß-oxidation regulators, PGC-1α (P < 0.05), PDK2 (P < 0.01), and PDK4 (P < 0.01) mRNA expression in singleton and twin fetuses at 135-138 days gestation. In singletons, there was also lower hepatic PDK4 (P < 0.01), CPT-1 (P < 0.01), and PKCζ (P < 0.01) protein abundance in the PCUN and PIUN groups and a lower protein abundance of PDPK-1 (P < 0.05) in the PCUN group. Interestingly, in twins, the hepatic protein abundance of p-AMPK (Ser(485)) (P < 0.01), p-PDPK-1 (Ser(41)) (P < 0.05), and PKCζ (P < 0.05) was higher in the PCUN and PIUN groups, and hepatic PDK4 (P < 0.001) and CPT-1 (P < 0.05) protein abundance was also higher in the PIUN twin fetus. We also found that the expression of a number of microRNAs was altered in response to PCUN or PIUN and that there is evidence that these changes may underlie the changes in the protein abundance of key regulators of hepatic fatty acid ß-oxidation in the PCUN and PIUN groups. Therefore, embryo number and the timing of maternal undernutrition in early pregnancy have a differential impact on hepatic microRNA expression and on the factors that regulate hepatic fatty acid oxidation and lipid synthesis.

Impact of periconceptional and preimplantation undernutrition on factors regulating myogenesis and protein synthesis in muscle of singleton and twin fetal sheep.

In this study, we determined the effect of maternal undernutrition in the periconceptional (PCUN: ~80 days before to 6 days after conception) and preimplantation (PIUN: 0-6 days after conception) periods on the mRNA and protein abundance of key factors regulating myogenesis and protein synthesis, and on the relationship between the abundance of these factors and specific microRNA expression in the quadriceps muscle of singleton and twin fetal sheep at 135-138 days of gestation. PCUN and PIUN resulted in a decrease in the protein abundance of MYF5, a factor which determines the myogenic lineage, in singletons and twins. Interestingly, there was a concomitant increase in insulin-like growth factor-1 mRNA expression, a decrease in the protein abundance of the myogenic inhibitor, myostatin (MSTN), and an increase in the mRNA and protein abundance of the MSTN inhibitor, follistatin (FST), in the PCUN and PIUN groups in both singletons and twins. These promyogenic changes may compensate for the decrease in MYF5 protein abundance evoked by early embryonic undernutrition. PCUN and PIUN also increased the protein abundance of phosphorylated eukaryotic translation initiation factor binding protein 1 (EIF4EBP1; T70 and S65) in fetal muscle in singletons and twins. There was a significant inverse relationship between the expression of miR-30a-5p, miR-30d-5p, miR-27b-3p, miR106b-5p, and miR-376b and the protein abundance of mechanistic target of rapamycin (MTOR), FST, or MYF5 in singletons or twins. In particular, the expression of miR-30a-5p was increased and MYF5 protein abundance was decreased, in PCUN and PIUN twins supporting the conclusion that the impact of PCUN and PIUN is predominantly on the embryo.

Maternal obesity or weight loss around conception impacts hepatic fatty acid metabolism in the offspring.

OBJECTIVE: To determine the impact of maternal obesity or weight loss during the periconceptional period on programming of lipid metabolism in the liver of the offspring. METHODS: An embryo transfer model was used to investigate the effects of exposure to either maternal obesity and/or weight loss before and for 1-week post-conception on the abundance of key molecules regulating hepatic fatty acid oxidation and lipid synthesis in the 4-month-old lamb. RESULTS: Periconceptional maternal obesity resulted in decreased hepatic PPARα, PGC1α and GCN5 abundance and increased hepatic SIRT1 and AMPKα1, AMPKα2 and SREBP1 abundance in the offspring. Maternal weight loss in obese ewes did not ablate all of these effects of maternal obesity on hepatic metabolism in the lamb. Weight loss in normal weight ewes also resulted in decreased hepatic PGC1α and GCN5 and increased AMPKα2 abundance in the offspring. CONCLUSIONS: Exposure of the oocyte/embryo to either maternal obesity or weight loss during the periconceptional period has long term consequences for hepatic lipid metabolism. These findings highlight the sensitivity of the early embryo to maternal nutrition and the need for dietary interventions which maximize metabolic benefits and minimize metabolic costs for the next generation.

Impact of embryo number and maternal undernutrition around the time of conception on insulin signaling and gluconeogenic factors and microRNAs in the liver of fetal sheep.

This study aimed to determine whether exposure of the oocyte and/or embryo to maternal undernutrition results in the later programming of insulin action in the liver and factors regulating gluconeogenesis. To do this, we collect livers from singleton and twin fetal sheep that were exposed to periconceptional (PCUN; -60 to 7 days) or preimplantation (PIUN; 0-7 days) undernutrition at 136-138 days of gestation (term = 150 days). The mRNA and protein abundance of insulin signaling and gluconeogenic factors were then quantified using qRT-PCR and Western blotting, respectively, and global microRNA expression was quantified using deep sequencing methodology. We found that hepatic PEPCK-C mRNA (P < 0.01) and protein abundance and the protein abundance of IRS-1 (P < 0.01), p110ß (P < 0.05), PTEN (P < 0.05), CREB (P < 0.01), and pCREB (Ser(133); P < 0.05) were decreased in the PCUN and PIUN singletons. In contrast, hepatic protein abundance of IRS-1 (P < 0.01), p85 (P < 0.01), p110ß (P < 0.001), PTEN (P < 0.01), Akt2 (P < 0.01), p-Akt (Ser(473); P < 0.01), and p-FOXO-1 (Thr24) (P < 0.01) was increased in twins. There was a decrease in PEPCK-C mRNA (P < 0.01) but, paradoxically, an increase in PEPCK-C protein (P < 0.001) in twins. Both PCUN and PIUN altered the hepatic expression of 23 specific microRNAs. We propose that the differential impact of maternal undernutrition in the presence of one or two embryos on mRNAs and proteins involved in the insulin signaling and gluconeogenesis is explained by changes in the expression of a suite of specific candidate microRNAs.