Chronic Undernutrition in Ovine Twin Pregnancies Abolishes Differences in Birth Weight Due to Sex: An Evaluation of the Role of Nutritional and Antioxidant Supplementation.

In twin pregnancies of discordant sex, the male fetus grows larger than the female co-twin. Our study aimed to determine the effect of the sex of co-twins on lambs' birth weight in ovine pregnancies developed under natural undernourishment. Additionally, we investigated whether the nutritional and/or antioxidant supplementation provided to ewes during pregnancy could modulate the potential effects associated with the sex of co-twins. Ninety-six birth records of twin pregnancies of sheep grazing the natural Patagonian prairies were analyzed. The animals were divided into four groups: control (no supplementation), N (concentrate supplementation, 100% NRC), A (antioxidant supplementation), and NA (concentrate + antioxidant supplementation). Supplementation occurred from day 35 of gestation onwards until lambing. There were no differences in female or male birth weight in the control undernourished group. However, in group N, females or males with sex-discordant co-twins had a higher birth weight than did those with co-twins of the same sex. Group A males with female co-twins had a higher birth weight compared to males whose co-twins were also males. In NA lambs, males had a higher birth weight compared to females, regardless of their co-twin's sex. Therefore, chronic undernutrition abolished the differences in birth weight due to fetal sex. Restoring maternal nutrition or antioxidant supplementation tends to normalize birth weight and restore the differences between females and males. This effect is enhanced with the combined supplementation of concentrated food and antioxidants.

Use of GnRH-Encapsulated Chitosan Nanoparticles as an Alternative to eCG for Induction of Estrus and Ovulation during Non-Breeding Season in Sheep.

This study is aimed at determining the reproductive performance of anestrous ewes treated with nanoencapsulated GnRH after a progesterone-based protocol for estrus induction was proposed as a way of replacing eCG. A total of sixty anestrous, multiparous, non-lactating Barki ewes were randomly allocated into three homogenous groups and subjected to a CIDR-based estrus induction protocol. The first group (eCG) received an intramuscular (i.m.) injection of 350 IU of eCG at CIDR removal. The second (LNGnRH) and third (HLNGnR) groups received either 25 µg or 50 µg of encapsulated GnRH nanoparticles by the i.m. route in the form of spherical GnRH-encapsulated chitosan-TPP nanoparticles (which were 490.8 nm and had a 13.6 mV positive charge) 48 h after CIDR removal. Follicular dynamics, estrous behavior, luteal activity, and pregnancy outcomes were evaluated. Three days after CIDR removal, the number of large follicles increased by similar amounts in the LNGnRH and eCG groups and were significantly higher in both groups than in the HNGnRH group. However, no differences were observed in the numbers and diameters of CLs among the experimental groups and, on the other hand, treatment with HNGnRH significantly increased blood serum progesterone levels compared with eCG and LNGnRH. Treatment with HNGnRH increased conception, lambing, and fecundity rates (p < 0.05), with the trend of a higher litter size (p = 0.081) compared with eCG, whereas LNGnRH resulted in intermediate values. In conclusion, a dose of 50 µg of GnRH encapsulated in chitosan-TPP nanoparticles can be used as an alternative to eCG in progesterone-based estrus induction protocols in sheep.

Ovarian Response and Fertility after Short-Term Progestagen/eCG Treatments Are Compromised in Nulliparous Sheep during Non-Breeding Season.

The objective of this investigation was to determine the ovarian response, fertility, and prolificacy of nulliparous sheep when compared to multiparous sheep after a short-term (7 days) CIDR/eCG treatment which was administered during the non-breeding season. All the multiparous sheep, whereas only 54% of the nulliparous ewes, showed signs of estrus. However, 81.8% of the multiparous sheep and 100% of the nulliparous ewes ovulated. Fertility was also low after short-term progesterone treatments during the anestrous season in maiden sheep (30.8 vs. 72.7% in multiparous ewes). Such results indicate significant differences in the response to CIDR/eCG protocols for induction and synchronization of estrus and ovulation between nulliparous and multiparous sheep during the non-breeding season.

Efficiency of hCG for Inducing Resumption of Ovarian Cyclicity and Synchronized Ovulations during the Seasonal Anestrous in Sheep.

This research aimed to evaluate whether the administration of hCG at the time of removal of a progesterone device may be effective at inducing estrus and ovulations in sheep during the natural seasonal anestrous, by comparing reproductive outputs (induction and duration of estrus, follicle development, ovulation, ovulation rate, and pregnancy rates) in ewes treated with eCG or only saline solution at the time of CIDR removal. Whereas results demonstrated no response in the control non-treated group, the largest rate of narrowly synchronized estrus signs and ovulations occurred in the eCG-group. The administration of hCG was effective at inducing estrus, promote follicular growth and a delayed yet significant ovulation (>84%) on day 10 after CIDR removal; moreover, an increased embryonic implantation rate was also observed. Moreover, if the hCG ewes remain exposed to active males for some days, said strategy could be adopted, aiming to induce pregnancies by natural mating in a short period of time.

The Use of hCG for Inducing Ovulation in Sheep Estrus Synchronization Impairs Ovulatory Follicle Growth and Fertility.

Currently, there is an intense effort to find an alternative hormone to eCG to induce ovulation after estrus synchronization treatments in sheep. One of the proposed alternatives is based on the use of hCG, but the results are controversial since fertility rates are commonly affected. The present study aims to evaluate, therefore, the adequacy of hCG in protocols for the synchronization of estrus and ovulation. Ovarian follicle dynamics, occurrence of estrus behavior and subsequent ovulation, quality of corpora lutea, and pregnancy rate after controlled natural mating were assessed in two consecutive trials. The findings indicate that the low fertility rates reported for the protocols based on the administration of hCG for inducing ovulation during estrus synchronization in sheep may be related to a high occurrence of abnormal follicular growth patterns, disturbances, and retardments of ovulation and concomitant formation of follicular cysts in the treated females. These results preclude their practical application to induce ovulation concomitantly to estrous synchronization treatments.

A Simplified Iohexol-Based Method to Measure Renal Function in Sheep Models of Renal Disease.

Sheep are highly adequate models for human renal diseases because of their many similarities in the histology and physiology of kidney and pathogenesis of kidney diseases. However, the lack of a simple method to measure glomerular filtration rate (GFR) limits its use as a model of renal diseases. Hence, we aimed to develop a simple method to measure GFR based on the plasma clearance of iohexol by assessing different pharmacokinetic models: (a) CL2: two-compartment (samples from 15 to 420 min; reference method); (b) CL1: one-compartment (samples from 60 to 420 min); (c) CLlf: CL1 adjusted by a correction formula and (d) SM: simplified CL2 (15 to 300 min). Specific statistics of agreement were used to test the models against CL2. The agreement between CL1 and CL2 was low, but both CL1f and SM showed excellent agreement with CL2, as indicated by a total deviation index of ~5-6%, a concordance correlation of 0.98-0.99% and a coverage probability of 99-100%, respectively. Hence, the SM approach is preferable due to a reduced number of samples and shorter duration of the procedure; two points that improve animal management and welfare.

Use of GnRH for Synchronization of the Follicular Wave in Assisted Reproductive Technologies in Sheep: A Preliminary Study.

The present study aimed to set up a short-term protocol for synchronization of follicular wave emergence in sheep, concomitant with estrus synchronization, which would improve ovarian response in assisted reproductive technologies. Administration of a single GnRH dose, concomitant with the insertion of a progesterone-loaded CIDR device, caused regression of gonadotrophin-dependent follicles ≥4 mm in all the GnRH-treated sheep and in around 80% of the controls treated only with CIDR (p < 0.05). Similar percentages of ewes lost all follicles (around 70%) or only the largest one (around 30%) in both groups. Hence, 54.1% and 70% of the sheep lost all large follicles and initiated a new follicular wave in the control and GnRH groups, respectively (p < 0.05). The remaining sheep showed follicles that were still not dependent of luteinizing hormone (LH). So, in fact, all the sheep had non-dominant follicles after treatment. In conclusion, a treatment including GnRH at CIDR insertion would offer a time- and cost-efficient protocol for inducing follicular turnover and synchronizing a new follicular wave at any stage of the estrous cycle.

Comparative efficiency of oestrus synchronization in sheep with progesterone/eCG and progesterone/GnRH during breeding and non-breeding season.

The present study compared the occurrence of oestrus behaviour and ovulation in response to the insertion of CIDR devices plus a classical treatment with equine chorionic gonadotrophin (eCG; single dose at CIDR removal) or alternative treatments with gonadotrophin-releasing hormone (GnRH, either in a single dose at 56 hr after CIDR removal, or in one dose at CIDR insertion and another dose at 56 hr after CIDR removal). The appearance of oestrus behaviour during reproductive season ranged between 84% and 95% and all females showing oestrus signs had subsequent ovulations. The response, during seasonal anoestrus, was similar in the group treated with eCG, but less than half of the females in the groups treated with GnRH showed oestrus signs in response to the treatment, although more than 80% of them showed resumption of ovulatory activity after the treatment. In conclusion, protocols based on GnRH administration offer similar yields to eCG-based protocols during the reproductive season but occurrence of oestrus in response to GnRH-based treatments is highly compromised during seasonal anoestrus.

Use of Propylene-Glycol as a Cosolvent for GnRH in Synchronization of Estrus and Ovulation in Sheep.

The foreseen shortage of eCG for estrus synchronization in sheep makes necessary the development of alternative protocols. The aim of the present work was to evaluate the reproductive response of sheep in breeding season to the administration of GnRH using propylene-glycol as a cosolvent and the subcutaneous route for slowing and extending the release of GnRH, as well as the most adequate timing for such administration. In the present study, protocols based on a short-term CIDR treatment and a single subcutaneous dose of GnRH in propylene-glycol at 36 h after CIDR removal induced a similar ovarian response to protocols based on administration of eCG at CIDR removal or intramuscular GnRH in distilled water at 56 h after. In such protocol, 80% of the animals developed estrus in a narrow timing (75% between 36 and 48 h after CIDR removal), and all of them also ovulated in a narrow window (87.5% between 72 and 76 h after CIDR removal, with 62.5% between 72 and 76 h) and showed a similar ovulation rate and plasma progesterone concentrations at the induced estrous cycle. Hence, administration of GnRH in propylene-glycol may constitute an alternative to traditional protocols based on the administration of eCG.

Seventy years of progestagen treatments for management of the sheep oestrous cycle: where we are and where we should go.

Management of the ovine oestrous cycle is mainly based on the use of exogenous hormones to mimic or enhance (progesterone and its analogues) or manipulate (prostaglandin F2α and its analogues) the activity of the corpus luteum, combined with the application of other hormones mimicking the pituitary secretion of gonadotrophins (e.g. equine chorionic gonadotrophin). These protocols have been applied without major change for decades but, now, there are two reasons to reconsider them: (1) our greatly improved knowledge of the dynamics of ovarian physiology, following the application of transrectal ultrasonography, indicates that modification of the protocols may improve fertility yields and (2) increasing concerns about animal health and welfare, food safety and the environmental impact of the treatments, as evidenced by public opinion and therefore market forces. Here, we offer an overview of these issues, introduce an updated protocol and suggest ways for future improvements to the protocols.

Placental oxygen transfer reduces hypoxia-reoxygenation swings in fetal blood in a sheep model of gestational sleep apnea.

Obstructive sleep apnea (OSA), characterized by events of hypoxia-reoxygenation, is highly prevalent in pregnancy, negatively affecting the gestation process and particularly the fetus. Whether the consequences of OSA for the fetus and offspring are mainly caused by systemic alterations in the mother or by a direct effect of intermittent hypoxia in the fetus is unknown. In fact, how apnea-induced hypoxemic swings in OSA are transmitted across the placenta remains to be investigated. The aim of this study was to test the hypothesis, based on a theoretical background on the damping effect of oxygen transfer in the placenta, that oxygen partial pressure (Po2) swings resulting from obstructive apneas mimicking OSA are mitigated in the fetal circulation. To this end, four anesthetized ewes close to term pregnancy were subjected to obstructive apneas consisting of 25-s airway obstructions. Real-time Po2 was measured in the maternal carotid artery and in the umbilical vein with fast-response fiber-optic oxygen sensors. The amplitudes of Po2 swings in the umbilical vein were considerably smaller [3.1 ± 1.0 vs. 21.0 ± 6.1 mmHg (mean ± SE); P < 0.05]. Corresponding estimated swings in fetal and maternal oxyhemoglobin saturation tracked Po2 swings. This study provides novel insights into fetal oxygenation in a model of gestational OSA and highlights the importance of further understanding the impact of sleep-disordered breathing on fetal and offspring development.NEW & NOTEWORTHY This study in an airway obstruction sheep model of gestational sleep apnea provides novel data on how swings in oxygen partial pressure (Po2) translate from maternal to fetal blood. Real-time simultaneous measurement of Po2 in maternal artery and in umbilical vein shows that placenta transfer attenuates the magnitude of oxygenation swings. These data prompt further investigation of the extent to which maternal apneas could induce similar direct oxidative stress in fetal and maternal tissues.

Onset of oestrus and periovulatory events in sheep exposed to 5 and 14 days of CIDR treatment with and without eCG.

The present study supports that 5-day short-term CIDR treatments without administration of eCG are equally effective for inducing oestrus behaviour, preovulatory LH discharge and ovulation in sheep than classical protocols based on 14-day treatments plus eCG at CIDR withdrawal. However, the implementation of a 5-day protocol without eCG for fixed-time artificial insemination would be adapted to a later timing of ovulation (p < .05).

Maternal Metabolic Demands Caused by Pregnancy and Lactation: Association with Productivity and Offspring Phenotype in High-Yielding Dairy Ewes.

Pregnancy and lactation, especially when concurrent, create a rather metabolically demanding situation in dairy ruminants, but little is known about their effects on offspring phenotype and milk yield. Here, we evaluated the impact of pregnancy and lactation on the metabolic traits and productive performance of Lacaune dairy sheep and their offspring. Productive performance was measured in terms of milk yield, body weight (BW), body condition score (BCS), and size. Productivity was assessed during mid-pregnancy (75 ± 5 d) and late pregnancy (142 ± 4 d) and at 52 ± 5 d in the postpartum period. During pregnancy, high-yielding ewes had higher BW, BCS, plasma glucose, cholesterol, ß-OHB, and NEFA than low-yielding ewes, but lower levels of lactate and urea. High-yielding animals had lower BCS after lambing, but their lambs showed greater growth. Productivity during lactation was affected by ewe age and parity: Mature ewes (but not maiden sheep) whose BCS increased steeply during pregnancy yielded more milk in the subsequent lactation than those whose BCS did not increase. Lamb BW and size were positively associated with milk yield in the subsequent lactation. Mature ewes had higher yields than maiden sheep, and mature ewes with multiple pregnancies produced more milk than those with singleton pregnancies. Ewes with male singleton pregnancies also showed higher yield than those with female singletons. These results demonstrate that high-yielding dairy sheep, when appropriately fed and managed, can adequately cover the metabolic demands of pregnancy and high milk production (even when concurrent) without losing productivity.

Efficiency of CIDR-Based Protocols Including GnRH Instead of eCG for Estrus Synchronization in Sheep.

The present study examined, for meat sheep (Segureña breed; 2-5-years old, mean body score of 3.5 ± 0.5), the timings of onset of estrus behavior, preovulatory luteinizing hormone (LH) surge and ovulation, and the ovulation rate and fertility obtained after insertion of controlled internal drug release (CIDR) devices for 5 days plus treatment with equine chorionic gonadotrophin (eCG; single dose at CIDR removal, n = 19 ewes) or gonadotrophin-releasing hormone (GnRH, either in a single dose at 56 h after CIDR removal, group CIDR-GnRH, n = 19 ewes; or in one dose at CIDR insertion and another dose 56 h after CIDR removal, group GnRH-CIDR-GnRH, n = 19 ewes). In all the ewes, the appearance of estrus behavior ranged between 84% and 90% and all females showing estrus signs had subsequent preovulatory LH peaks and ovulations. Onset of these events was earlier in the CIDR-eCG group than in the CIDR-GnRH and GnRH-CIDR-GnRH groups (p < 0.05). These differences were mainly determined by the onset of estrus behavior, since timing and intervals of LH peak and ovulation were similar among treatments. In fact, the range of ovulations was narrower in the GnRH-CIDR-GnRH group, which suggests better synchronization of follicular growth (p < 0.05). In conclusion, protocols with two doses of GnRH offer similar yields to eCG protocols.

Influence of Maternal Factors (Weight, Body Condition, Parity, and Pregnancy Rank) on Plasma Metabolites of Dairy Ewes and Their Lambs.

Pregnancy and lactation are challenging states that affect maternal and lamb health. In Lacaune dairy sheep, we evaluated the impact of parity, pregnancy rank, and body condition on body weight and the condition of ewes and lambs in mid-pregnancy (75 ± 5 d), in late pregnancy (142 ± 4d), and postpartum (52 ± 5d pp). Maternal age was associated with initial decreases, followed by increases, in body weight and condition. After lambing, both mature and maiden ewes lost weight and body condition. Maternal indices of glucose, protein, and lipid metabolism were within physiological values during pregnancy, but postpartum values depended on maternal parity and pregnancy rank, with multiple-pregnant ewes showing a postpartum increase in glucose and maiden sheep a postpartum increase in plasma cholesterol concentration. Male lambs were heavier than female lambs at birth, and lambs born to mothers with higher body condition scores were heavier. Lambs born as singletons were heavier than those born in litters. Maternal age and pregnancy rank did not influence lamb metabolic indicators. Sex affected plasma concentrations of glucose, triglycerides, and cholesterol. Maternal metabolic indicators showed minimal effects on lamb phenotype. These results suggest that, when appropriately fed, dairy sheep can cover the metabolic demands of pregnancy and milk production, regardless of age and pregnancy rank.

Onset of estrus and preovulatory LH surge and ovulatory efficiency in sheep after short-term treatments with progestagen-sponges and progesterone-CIDRs.

The present study supports that short-term (7-days) protocols based on progestagen-impregnated sponges and progesterone-loaded CIDRs are equally effective to induce ovulatory response in sheep. There were no significant differences in the onset of estrus behavior (32.0 ± 6.0 and 33.8 ± 4.0 hr after device withdrawal for sponges and CIDRs, respectively; p > 0.05) and preovulatory LH discharge (5.1 ± 2.1 and 5.8 ± 3.3 hr after onset of estrus behavior for sponges and CIDRs, respectively; p > 0.05). These features are similar to previously described for classical long-term (12-14 days) treatments. Hence, short-term CIDR-based protocols may be implemented using the same time-intervals for insemination than sponge-based and long-term protocols.

Influence of Progesterone-Treatment Length and eCG Administration on Appearance of Estrus Behavior, Ovulatory Success and Fertility in Sheep.

The present study characterizes, for sheep, the occurrence and timing of the onset of estrus behavior and ovulation and the yields obtained (ovulation rate, progesterone secretion, and fertility) after Controlled Internal Drug Release (CIDR) insertion for five, six, seven, or fourteen days, with or without equine chorionic gonadotrophin (eCG) in ewes of the Segureña meat breed. All the treatments showed above 80% of females displaying estrus, but the onset of estrus was earlier and more synchronized when using eCG and, among groups with eCG, onset of estrus was earlier in the sheep treated for 14 days than in the short-term treatments (p < 0.05 for all). Administration of eCG after either short- or long-term treatments assured the occurrence of fertile ovulations in all the animals. Conversely, ovulatory success without eCG was found to be dependent on treatment length, with a high percentage of animals ovulating after five days of treatment (83.3%) and very low percentages after treatment for six or seven days (40% and 20%, respectively). Ovulation rate and progesterone secretion were similar among animals ovulating, but ovulation failures predetermined the fertility yields obtained in response to the treatments. Hence, the best results were found after treatment for 14 days plus eCG, and for 5 days without eCG (83.3 for both, p < 0.05 when compared to the other groups with different treatment lengths and with or without eCG).

Intravaginal Device-Type and Treatment-Length for Ovine Estrus Synchronization Modify Vaginal Mucus and Microbiota and Affect Fertility.

Induction and synchronization of estrus and ovulation in sheep is based on intravaginal progestagen-impregnated polyurethane sponges or progesterone-loaded silicon-based devices (CIDR), in either short- (6⁻7 days) or long-term (12⁻14 days) protocols. Bearing in mind that the use of intravaginal sponges in long-term protocols has been related to the presence of vaginitis at removal, we compared the effects of sponges and CIDRs, maintained during either 7 or 14 days, on vaginal features (characteristics of vaginal mucus discharge, pH and microbiota) and fertility under field conditions. Almost all the ewes treated with intravaginal sponges showed vaginal discharge at device withdrawal, which was purulent and/or bloody in around 15% and 80% of the females treated for 7 and 14 days, respectively. The vaginal pH and microbiota changed in both groups when compared to control sheep, especially in ewes treated for 14 days, which showed a pH value around 8 and a higher incidence of Salmonella spp. and Staphylococcus aureus. On the other hand, independently of the length of the treatment, only around 15⁻20% of the sheep treated with CIDRs evidenced vaginal discharge (p < 0.00005 when compared to sponge groups), and such discharge was scarce, clear, and showed no changes in vaginal pH and microbiota when compared to control sheep. Fertility yields were associated with vaginal features, being higher in both short-term treatments (75%) and the long-term CIDR-based treatment (70%) than in the long-term sponge-based treatment (45%).

Effects of short-term intravaginal progestagens on the onset and features of estrus, preovulatory LH surge and ovulation in sheep.

In the present study, there was a comparison among classical long-term progestagen (fluorogestone acetate) protocols for synchronization of estrus and ovulation (14 days; group FGA14, n = 9 ewes) and short-term protocols based on 7 days of progestagen treatment plus a dose of prostaglandin F2α at either insertion (PG-FGA7, n = 11) or removal (FGA7-PG, n = 12). There were no significant differences in the ovulation rate and progesterone secretion among treatments. The FGA7-PG group, however, had a similar percentage of ewes expressing estrous behavior than the group FGA14 (90.9 and 100%, respectively, with a trend for a lesser percentage in the PG-FGA7 group, 63.6%) and about 90% of the ewes in the FGA7-PG group had the preovulatory surge release of LH 8 h after the onset of estrous behavior. These features may be related to a greater number of preovulatory follicles during growing phases (P < 0.05) and a greater plasma estradiol concentration (P < 0.05) in this group than in the classical 14-day group, which suggest these are more functional preovulatory follicles. In conclusion, therefore, the use of the FGA7-PG treatment may favor efficiency of progestagen-based protocols for reproductive management.