Uterine tone: A neglected criterion for the selection of bovine embryo transfer recipients.

This study evaluated the relationship between CL features assessed by ultrasound (luteal tissue area and blood flow, BF) or rectal palpation (size), uterine tone (UT), plasma progesterone (P4) concentration on Day 7 (D7) and subsequent pregnancy outcomes in bovine embryo recipients. A total of 163 cows and heifers were included in this study. The expected day of ovulation after the synchronization protocol was designated as D0. On D7, ovaries and uterus were examined by ultrasonography and rectal palpation, and subjective scores (1-3 scale) were assigned for CL size, area and BF, and for UT. Blood samples were collected for further P4 analysis. Each embryo recipient then received a grade I frozen-thawed in vivo-produced blastocyst, which was transferred to the uterine horn ipsilateral to the CL. Pregnancy diagnosis was performed on D35, and the results were retrospectively compared with the assigned scores for CL and UT. We observed a significant (p < .02) interaction between CL size and UT, with a progressive increase in the likelihood of pregnancy for recipients bearing a large CL among those with turgid UT. Ultrasound scoring of the CL using B-mode and Doppler-mode did not significantly predict pregnancy rates on D35 (p < .6 and p < .5, respectively). However, logistic regression analysis revealed a trend towards a quadratic effect (p < .08 and p < .06) indicating that the probability of pregnancy varied according to the area of luteal tissue and P4 concentrations, respectively. No significant (p > .05) association was found between the probability of pregnancy and the BF area of the CL. In summary, UT before embryo transfer may reflect successful recipient synchronization. Elevated P4 levels, assessed by CL size, may offset uterine contractility, mitigating adverse effects. Additionally, the CL area may be more important than its vascularization area when evaluating recipients D7 after ovulation.

Effect of prostaglandin treatment on the estrus behaviour, follicular and luteal morphometry and serum hormone profile in sub-estrus buffaloes during non-breeding season.

Sub-estrus buffaloes do not exhibit estrus signs despite being cyclic contributing to extended service periods and inter-calving intervals causing significant economic loss. The present study described the effect of synthetic prostaglandin (PGF2α) on estrus behaviour, follicular and luteal morphometry, and serum estradiol (E2) and progesterone (P4) profile in sub-estrus buffaloes during the non-breeding season. The incidence of sub-estrus was 38.4% during the non-breeding season. The sub-estrus buffaloes (n = 33) were divided into two groups, viz., Control (n = 16) and PGF2α treatment (Inj. Cloprostenol 500 µg, i.m., n = 17). Estrus induction response was significantly greater in the treatment (100 vs. 18.75%, p < .001), and a relatively greater proportion of animals conceived in the treatment group (29.41 vs. 6.25%, p = .08). The time elapsed to induction of estrus and insemination following treatment was significantly lower in the treatment group than control. A significant increment in the follicle diameter (9.72 ± 0.45 vs. 13.00 ± 0.45 mm, P < .0001) and serum estradiol (E2) concentration (66.01 ± 11.92 vs. 104.9 ± 13.21 pg/mL, p = .003) observed at the post-treatment period in the PGF2α treatment group. At the same time, CL diameter was reduced significantly at a higher regression rate in the PGF2α treated buffaloes than those of control. Of the responded buffaloes, only 30% showed high-intensity estrus attributed to the expulsion of cervico-vaginal mucus (CVM), uterine tonicity, micturition, and mounting response by a teaser bull. From this study, it can be concluded that the administration of PGF2α could induce estrus in the sub-estrus buffaloes during the non-breeding season. Behavioural changes, along with sonographic observation of POF, regressing CL, and serum E2 and P4 concentration would be useful to determine the right time of insemination in sub-estrus buffaloes during non-breeding season.

Effect of prostaglandin alone and in combination with trace minerals on the follicular and luteal dynamics, estrus response and pregnancy in sub-estrus buffaloes (Bubalus bubalis).

Sub-estrus is a condition when buffaloes do not display behavioural estrus signs, despite being in estrus and causes a delay in conception and increases the service period. The present study describes the effect of synthetic prostaglandin (PGF2α) alone and in combination with trace minerals on the follicular and corpus luteum (CL) dynamics, serum estradiol (E2) and progesterone (P4) concentration correlating estrus response and pregnancy outcome in sub-estrus buffaloes during the breeding season. A total of 50 sub-estrus buffaloes, identified through ultrasonography (USG) examination, were randomly allocated into three groups, viz. T1 (Synthetic PGF2α, Inj. Cloprostenol 500 µg, i.m, n = 17), T2 (Synthetic PGF2α + Trace mineral supplementation, Inj. Stimvet 1 mL/100 kg body weight, i.m., n = 17) and control (untreated; n = 16). Following treatment, 100% of sub-estrus buffaloes were induced estrus in the T1 and T2 groups, while only 18.75% were induced in the control. The CL diameter and serum P4 concentration were significantly lower at post-treatment, whereas the pre-ovulatory follicle (POF) size and serum E2 concentration were significantly higher in the T1 and T2 groups as compared to the control (p < .05). The buffaloes of the T2 group had a greater proportion of moderate intensities estrus than those of T1. Moreover, the proportion of buffaloes conceived in the T1 and T2 were 41.2% and 52.95%, respectively. The larger POF diameter and higher serum E2 concentration were associated with intense intensity estrus and higher conception rate (66.7%) in sub-estrus buffaloes. Similarly, CL regression rate, POF size and serum E2 concentration were relatively higher in the buffaloes conceived as compared to those not conceived. It is concluded that synthetic PGF2α in combination with trace minerals induces moderate to intense intensities estrus in a greater proportion of sub-estrus buffaloes and increases the conception rate during the breeding season. Moreover, behavioural estrus attributes correlating follicle and luteal morphometry, serum E2 and P4 concentration could be used to optimise the breeding time for augmenting the conception rate in sub-estrus buffaloes.

Enhancement of progesterone biosynthesis via kisspeptin stimulation: Upregulation of steroidogenic transcripts and phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in the buffalo luteal cells.

The presence of Kisspeptin (Kp) and its receptors in the corpus luteum (CL) of buffalo has recently been demonstrated. In this study, we investigated the role of Kp in the modulation of progesterone (P4) synthesis in vitro. The primary culture of bubaline luteal cells (LCs) was treated with 10, 50, and 100 nM of Kp and Kp antagonist (KpA) alongside a vehicle control. The combined effect of Kp and KpA was assessed at 100 nM concentration. Intracellular response to Kp treatment in the LCs was assessed by examining transcript profiles (LHR, STAR, CYP11A1, HSD3B1, and ERK1/2) using quantitative polymerase chain reaction (qPCR). In addition, the immunolocalization of ERK1/2 and phosphorylated ERK1/2 (p-ERK1/2) in the LCs was studied using immunocytochemistry. Accumulation of P4 from the culture supernatant was determined using enzyme-linked immunosorbent assay (ELISA). The results indicated that LCs had a greater p-ERK1/2 expression in the Kp treatment groups. A significant increase in the P4 concentration was recorded at 50 nM and 100 nM Kp, while KpA did not affect the basal concentration of P4. However, the addition of KpA to the Kp-treated group at 100 nM concentration suppressed the Kp-induced P4 accumulation into a concentration similar to the control. There was significant upregulation of ERK1/2 and CYP11A1 expressions in the Kp-treated LCs at 100 nM (18.1 and 37fold, respectively, p < 0.01). However, the addition of KpA to Kp-treated LCs modulated ERK1/2, LHR, STAR, CYP11A1, and HSD3B1 at 100 nM concentration. It can be concluded that Kp at 100 nM stimulated P4 production, while the addition of KpA suppressed Kp-induced P4 production in the buffalo LCs culture. Furthermore, an increment in p-ERK1/2 expression in the LCs indicated activation of the Kp signaling pathway was associated with luteal steroidogenesis.

Role of chemokines in regulating luteal and uterine functions in pregnant cows.

Pregnancy is intricately regulated by the interactions between various bioactive substances secreted by the conceptus, uterus, and corpus luteum (CL). Interferon-τ, synthesized and secreted by the conceptus, plays a central role in the interaction mechanism of maternal recognition in cows. Chemokines, chemotaxis mediators that are primarily secreted by immune cells, regulate various reproductive responses in various species. Although there are scattered reports on the potential roles of chemokines in the bovine CL and the uterus during the estrous cycle, there is little information on chemokines in these organs during pregnancy. Therefore, in this review, we discuss the possible physiological roles of chemokines in the CL and uterus of pregnant cows, focusing on our recent findings on chemokines and changes in their receptor expression in the CL and endometrium of cows at some stages of pregnancy.

Uteroovarian pathway for embryo-empowered maintenance of the corpus luteum in farm animals.

The first luteal response to pregnancy in farm animals at 12-18 days after ovulation involves maintenance of the corpus luteum (CL) if pregnancy has occurred. In most common farm species, regression of the CL results from production of a luteolysin (PGF2α) by the nongravid uterus, and maintenance of the CL involves the production of an antiluteolysin (PGE2) by the gravid uterus and conceptus. The proximal component of a unilateral pathway from a uterine horn to the adjacent CL for transport of PGF2α and PGE2 is the uterine venous and lymphatic vessels and the distal component is the ovarian artery. The mechanisms for venolymphatic arterial transport of PGF2α and PGE2 from a uterine horn to the adjacent CL ovary and transfer of each prostaglandin through the walls of the uteroovarian vein and ovarian artery occur by similar mechanisms probably as a consequence of similarities in molecular structure between the two prostaglandins. Reported conclusions or interpretations during the first luteal response to pregnancy in sows and ewes are that PGE2 increases in concentration in the uteroovarian vein and ovarian artery and counteracts the negative effect of PGF2α on the CL. In cows, treatment with PGE2 increases circulating progesterone concentrations and prevents spontaneous luteolysis and luteolysis induced by estradiol, an intrauterine device, or PGF2α. The prevailing acceptance that interferon tau is the primary factor for maintaining the CL during early pregnancy in ruminants will likely become tempered by the increasing reports on PGE2.

Laterality in roe deer embryos implantation.

Female reproductive success is one of the most important life-history traits to be monitored when determining population dynamics in free-ranging ungulates. Several studies have described how phenotypic characteristics of the mother, climatic conditions, population status, and habitat can impact on potential reproductive output in wild ungulates. However, little is known regarding the internal, physiological factors, that may account for differences in implantation rates. The present study investigated the differences in implantation rates and site on the basis of site and number of ovulations through the examination of about 3000 intact uteri collected from pregnant roe deer does (Capreolus capreolus). Although ovulation occurs with the same frequency in the left and right ovary, we revealed a higher frequency of embryos implantation in the left uterine horn in odd litter size, demonstrating that embryos can migrate between the uterine horns. In our study, a greater proportion of reproductive wastage was associated to females with three and four corpora lutea and interestingly, in relation to the site of ovulation, the percentage of corpora lutea that did not correspond to a fetus was higher in the right ovary than in the left one (73.2% vs. 26.8%). Our research described for the first time the absence of laterality in ovulation and the presence of laterality in implantation in roe deer, thus laying the foundations for in-depth studies about the functionality of this uterine side and for comparisons with populations located in other geographical areas to understand whether it is a widespread phenomenon or a local adaptation.

miR-665 overexpression inhibits the apoptosis of luteal cells in small ruminants suppressing HPGDS.

Evidence has shown that microRNA-665 (miR-665) is highly expressed in the mid-luteal phase compared with the early and end-luteal phase of the corpus luteum (CL) life cycle. However, whether miR-665 is a positive regulator of the life span of the CL is still unknown. The objective of this study is to explore the effect of miR-665 on the structural luteolysis in the ovarian CL. In this study, the targeting relationship between miR-665 and hematopoietic prostaglandin synthase (HPGDS) was firstly verified by dual luciferase reporter assay. Then, quantitative real-time PCR (qRT-PCR) was used to detect the expression of miR-665 and HPGDS in luteal cells. Following miR-665 overexpression, the apoptosis rate of the luteal cells was determined using flow cytometry; B-cell lymphoma-2 (BCL-2) and caspase-3 mRNA and protein were measured using qRT-PCR and Western blot (WB) analysis. Finally, the DP1 and CRTH2 receptors of PGD2, a synthetic product of HPGDS, were localized using immunofluorescence. Results confirmed that HPGDS was a direct target gene of miR-665, and miR-665 expression was negatively correlated with HPGDS mRNA expression in luteal cells. Meanwhile, after miR-665 was overexpressed, the apoptotic rate of the luteal cells showed a significant decrease (P < 0.05) and this was accompanied by elevated expression levels of anti-apoptotic factor BCL-2 mRNA and protein and decreased expression levels of apoptotic factor caspase-3 mRNA and protein (P < 0.01). Moreover, the immune fluorescence staining results showed that the DP1 receptor was also significantly decreased (P < 0.05), but the CRTH2 receptor was significantly increased (P < 0.05) in luteal cells. Overall, these results indicate that miR-665 reduces the apoptosis of luteal cells via inhibiting caspase-3 expression and promoting BCL-2 expression, and the biological function of miR-665 may be attributed to its target gene HPGDS which regulates the balance of DP1 and CRTH2 receptors expression in luteal cells. As a consequence, this study suggests that miR-665 might be a positive regulator of the life span of the CL rather than destroy the integrity of CL in small ruminants.

Effect of early administration of progesterone on the function of the corpus luteum of llamas.

The aim of the present study was to evaluate if early administration of progesterone immediately after ovulation affects corpus luteum lifespan in llamas. Female llamas (n = 16) were induced to ovulate by Buserelin injection in the presence of an ovulatory follicle (Day 0). On Day 2, ovulation was confirmed and animals were randomly divided into two groups: treated animals (n = 8) received an intravaginal device containing 0.3 g of progesterone from Day 2 to Day 6 post-induction of ovulation and control group (n = 8) received a device with 0 g of progesterone. Blood samples were collected daily to determine plasma progesterone concentration and transrectal ultrasonographies were performed from Day 7 to Day 12 post-induction of ovulation. Mean maximum diameter of the corpus luteum was significantly lower and was reached before in the treated group than in the control group. The mean highest plasma progesterone concentration and the day that concentration was achieved were similar between groups. However, mean plasma progesterone concentration was significantly higher in the treated group than in the control group on Days 3 and 4 and lower on Days 8 and 9 post-induction of ovulation. The day that plasma progesterone concentration returns to 1 ng/ml differed between groups, occurring earlier in the treated group. In conclusion, the early increase of plasma progesterone concentration during the luteal phase, promoted the premature activation of the luteolytic process affecting corpus luteum function in llamas as it was previously reported in other species.

In vitro effects of PPAR gamma ligands on gene expression in corpus luteum explants in non-pregnant pigs - Transcriptome analysis.

The corpus luteum (CL) is a temporary endocrine structure in the female ovaries that develops cyclically in mature females during luteinization. This study aimed to determine the in vitro effects of peroxisome proliferator-activated receptor gamma (PPARγ) ligands on the transcriptomic profile of the porcine CL in the mid- and late-luteal phase of the estrous cycle using RNA-seq technology. The CL slices were incubated in the presence of PPARγ agonist - pioglitazone or antagonist - T0070907. We identified 40 differentially expressed genes after treatment with pioglitazone and 40 after treatment with T0070907 in the mid-luteal phase as well as 26 after pioglitazone and 29 after T0070907 treatment in the late-luteal phase of the estrous cycle. In addition, we detected differences in gene expression between the mid- and late-luteal phase without treatment (409 differentially expressed genes). This study revealed a number of novel candidate genes that may play a role in controlling the function of CL by regulating signaling pathways related to ovarian steroidogenesis, metabolic processes, cell differentiation, apoptosis, and immune responses. These findings become a basis for further studies to explain the mechanism of PPARγ action in the reproductive system.

Expression and regulation of visfatin/NAMPT in the porcine corpus luteum during the estrous cycle and early pregnancy.

Visfatin/NAMPT creates a hormonal link between energy metabolism and female reproduction. A recent study documented visfatin expression in the ovary and its action on follicular cells; however, the expression of visfatin in luteal cells is still unknown. The aim of this study, therefore, was to investigate the transcript and protein expression of visfatin as well as its immunolocalization in the corpus luteum (CL) and to examine the involvement of extracellular signal-regulated kinases (ERK1/2) in the regulation of visfatin level in response to LH, insulin, progesterone (P4), prostaglandin E2 (PGE2) and F2α (PGF2α). Corpora lutea were harvested from gilts on days 2-3, 10-12 and 14-16 of the estrous cycle and on days 10-11, 12-13, 15-16 and 27-28 of pregnancy. The current study demonstrated that visfatin expression depends on hormonal status related to the phase of the estrous cycle or early pregnancy. Visfatin was immunolocalized to the cytoplasm of small and large luteal cells. Moreover, visfatin protein abundance was increased by P4, and decreased by both prostaglandins, while LH and insulin have modulatory effects, depending on the phase of the cycle. Interestingly, LH, P4 and PGE2 effects were abolished in response to the inhibition of ERK1/2 kinase. Thus, this study demonstrated that expression of visfatin in the porcine CL is determined by the endocrine status related to the estrous cycle and early pregnancy and by the action of LH, insulin, P4 and prostaglandins via activation of the ERK1/2 pathway.

Enhanced progesterone support during stimulated cycles of transvaginal follicular aspiration improves bovine in vitro embryo production.

The in vitro production (IVP) of cattle embryos requires that germinal-vesicle stage oocytes undergo a period of maturation in vitro prior to fertilization and culture to the blastocyst stage. Success of IVP in taurine cattle is enhanced following ovarian stimulation prior to oocyte retrieval (OPU), particularly if preceded by a short period of FSH withdrawal ('coasting'). However, evidence regarding the importance of progesterone (P4) support during OPU-IVP is equivocal. The current study, therefore, determined the effects of increased peripheral P4 concentrations during FSH-stimulated ('coasted') cycles of OPU. Progesterone support was provided by either an active corpus luteum (CL) and/or one of two intravaginal P4 releasing devices (i.e., CIDR® [1.38 g P4] or PRID® Delta [1.55 g P4]). Expt. 1 established an initial estrus prior to OPU, allowing CL formation (single luteal phase) spanning the first two of five cycles of OPU; the remaining three cycles were supported by either a CIDR® or PRID® Delta. Expt. 2 commenced with two cycles of dominant follicle removal (including prostaglandin F2α) undertaken seven days apart prior to six cycles of OPU. The absence of a CL meant that these cycles were supported only by a CIDR® or PRID® Delta. As each experiment involved several sequential cycles of OPU, the cumulative effects of device use on vaginal discharges were also assessed. Each experiment involved 10 sexually mature Holstein heifers. In the absence of a CL, peak plasma P4 concentrations were greater (P = 0.002) for the PRID® Delta (4.3 ± 0.22) than for the CIDR® (2.9 ± 0.22). In Expt. 1 there was an interaction (P < 0.05) between CL presence at OPU and P4 device on Day 8 blastocyst yields, indicating an effect of P4 device only when the CL was absent. The percentage hatching/hatched blastocysts of matured oocytes for the CIDR® and PRID® Delta was 44.3 ± 5.04 and 41.0 ± 5.40 in the presence, and 17.1 ± 3.48 and 42.2 ± 3.76 in the absence, of a CL (P = 0.018). Combined analyses of data from Expt. 1 and 2, when no CL was present, confirmed that Day 8 blastocyst yields were greater (P = 0.022) for the PRID® Delta than the CIDR®. Vaginal discharge scores were higher (P < 0.001) for the PRID® Delta than the CIDR® in Expt. 1 but not in Expt 2; however scores were low, did not increase with repeated use, and thus were deemed of no clinical or welfare concern. In conclusion, enhanced P4 support during FSH-stimulated cycles of OPU-IVP can improve in vitro embryo development.

Relationship between ovarian ultrasonographic findings on the seventh post-estrus day and plasma progesterone concentration, nutritional metabolic factors, and pregnancy outcome in dairy cows.

To improve the accuracy of ultrasonographic assessment of luteal function, we investigated the relationship between ovarian ultrasonographic findings on Day 7 (Day 1 = ovulation) and plasma progesterone (P4) concentration, nutritional metabolic factors, and pregnancy outcome. A total of 47 spontaneous estrus events were investigated in 38 lactating Holstein cows (artificial insemination, n = 31; embryo transfer, n = 16). Transrectal ultrasonography was performed on Days 0 and 7 to measure the pre-ovulatory follicle area on Day 0 and the luteal tissue area (LTA), luteal blood flow area (LBF), relative LBF (rLBF) (= LBF/LTA), and dominant follicle area (DFA) on Day 7. Blood samples were collected on Day 7 to measure plasma P4, insulin-like growth factor-I (IGF-I), insulin, and metabolites. Plasma P4 concentration was positively correlated with LTA but was not associated with LBF or rLBF. Plasma P4 concentration was positively correlated with blood glucose and IGF-I and negatively correlated with blood urea nitrogen and free fatty acid, and no significant relationship was found between the ultrasonographic findings of the corpus luteum (CL) and these blood metabolites. Pregnant cows had smaller DFA than non-pregnant cows. In conclusion, LTA measurement can help predict plasma P4 concentration, but it was difficult to detect variations in plasma P4 concentration in relation to changes in energy status by evaluating the CL ultrasonographically. A combined assessment of CL and first-wave dominant follicle may be important in evaluating fertility.

Serum progesterone and oxytocinase, and endometrial and luteal gene expression in pregnant, nonpregnant, oxytocin, carbetocin and meclofenamic acid treated mares.

Our objectives were to examine changes in endometrial and luteal gene expression during estrus, diestrus, pregnancy and treatments to induce luteolysis and putatively induce luteostasis. Groups were: Diestrus (DIEST), Estrus (ESTR), Pregnant (PREG), Oxytocin (OXY), Carbetocin (CARB), and Meclofenamic acid (MFA). Blood was obtained from day (D)12 to D15 for measurement of oxytocinase, also referred to as leucyl-cysteinyl aminopeptidase (LNPEP) and progesterone. Luteal biopsies were obtained on D12 and D15 and an endometrial biopsy on D15. Real-time RT-PCR was performed for the following genes: PGR, ESR1, OXTR,OXT, LNPEP, PTGS2, PTGFR, PLA2G2C, PTGES, SLC2A4, and SLC2A1. Regarding serum LNPEP, PREG and OXY (p-value<0.001) had higher concentrations than DIEST mares. Endometrial PTGES expression was higher (p-value <0.04) in DIEST, PREG and OXY than other groups. Endometrium from ESTR had increased expression of OXT (p-value < 0.02) compared to MFA and OXY mares. Carbetocin treatment: decreased serum progesterone and LNPEP; increased endometrial PLA2G2C; decreased endometrial PTGES; and decreased luteal aromatase and PTGES. Treatment with MFA: decreased endometrial PLA2G2C, increased endometrial PTGES; and resulted in less OXTR and OXT luteal abundance on D12 compared to D15. Endometrial and luteal expression of LNPEP is affected by physiologic stage and treatment and is involved in luteal function and pregnancy recognition pathways through effects on oxytocin and prostaglandin synthesis in the horse.

Expressions of ghrelin and GHSR-1a in the corpus luteum and the stimulatory effect of ghrelin on luteal function of pregnant sows.

Studies have shown that ghrelin played direct actions in ovarian function, but the direct role of ghrelin in corpus luteum (CL) of pregnant sows has remained obscure. The study aimed to examine the expressions of ghrelin and its functional receptor (GHSR-1a) in the CL of sows during pregnancy, and evaluate the role of ghrelin in CL function of pregnant sows. Immunohistochemistry analysis showed that ghrelin and GHSR-1a are both predominantly localized in the luteal cells of pregnant sows CL. Strong immunoreactivity for ghrelin and GHSR-1a is detected at days 20 (early) and 50 (middle), but weak immunoreactivity is observed at days 90 (late) post mating. Similarly, there is a significant effect of pregnant phase on the expression (mRNA and protein) of ghrelin and GHSR-1a in the CL, with higher levels at days 20 (early) and 50 (middle), and lower values at 90 (late) post mating. In vitro, treatments of luteal cells with ghrelin (from 0.01 to 10 ng/mL) are promoted cell viability and P4 secretion in a dose-dependent manner. Ghrelin is also accelerated the LH-induced P4 secretion in luteal cells. Moreover, ghrelin is induced the release and mRNA expression of LH, and increased the release of prostaglandin (PG)E2, but reduced the secretion of PGF2α in luteal cells. In conclusion, the presences of ghrelin and GHSR-1a in the porcine CL during pregnancy, and the stimulatory effect of ghrelin on luteal cells suggest positive regulation by ghrelin in CL function of pregnant sows.

Single dose of 300 IU hCG in the early luteal phase in superovulated ewes: Effects on corpora lutea, progesterone profile, and embryo recovery.

This study aimed to evaluate the effects of hCG treatment during the early luteal phase on ovarian function, progesterone profile, and embryo yield in superovulated ewes. Superovulated sheep were randomly assigned to receive 300 IU hCG i.m. (GhCG, n = 24) or not (GControl, n = 25) at 96 h after the removal of the progesterone (P4) device (D13). Non-surgical embryo recovery (NSER) was performed eight days after P4 withdrawal. Ultrasound evaluations were performed on D13, D14, D16, and D17. Blood samples were collected on D14, D16, and D17. Superovulation scores were recorded based on the number of corpora lutea (CL) as follows: 1 (≤ 2), 2 (3-5), 3 (6-8), and 4 (≥ 9). NSER efficiency, superovulation response, and luteal tissue area were similar in both groups (P > 0.05). Structural luteolysis tended to be higher in GControl (P = 0.07; 47.0 %) while functional luteolysis was similar in both groups (P > 0.05; 0.0 % and 5.9 %). The recovery rate was greater (P < 0.05) in GhCG (89.8 %) compared with GControl (71.0 %), with similar overall ova/embryo numbers observed for both groups (P > 0.05). GhCG showed a higher concentration of animals with a superovulatory response score of 4 (54.5 %; P < 0.05) compared with the lowest scores. Plasma progesterone on D16 was higher (P < 0.05) in GhCG ewes (11.1 ± 1.5 vs 6.9 ± 1.5 ng/mL). In conclusion, the hCG treatment improved circulating P4 and embryo recovery rate, tended to maintain luteal functionality, and thus constitutes an additional tool for improving embryo yield in superovulated ewes.

Differential expression and functional prediction of mRNA in the ovaries of Hanper sheep of high and low fecundity.

Hanper ewes that were either monotocous or polytocous provided ovarian follicles of diameter >3 mm in the follicular phase and, in the luteal phase, samples of corpora lutea that had developed from follicles of diameter >3 mm. Differentially expressed mRNAs (monotocous versus polytocous) were then identified, and their functions were predicted. Results showed that 1508 mRNAs were differentially expressed in the follicular phase, with 885 being in the luteal tissues. Those which were differentially expressed in the follicular phase were mainly involved in the regulation of the ferroptosis and lysosome signalling pathways, whereas, for the luteal tissue, the differentially expressed mRNAs were mainly involved in the regulation of steroid biosynthesis. Based on the results, it was inferred that these pathways could explain variations in the fecundity of sheep.

Expression patterns of chemokine (C-C motif) ligand 2, prostaglandin F2A receptor and immediate early genes at mRNA level in the bovine corpus luteum after intrauterine treatment with a low dose of prostaglandin F2A.

The present study evaluated expression patterns of chemokine (C-C motif) ligand 2 gene/Monocyte chemoattractant protein-1 gene (CCL2/MCP-1), prostaglandin F2 alpha receptor gene (PTGFR) and immediate early genes including nuclear receptor subfamily 4, group A, member 1 (NR4A1), early growth response 1 (EGR1) and FBJ murine osteosarcoma viral oncogene homolog (FOS) in cells of the bovine corpus luteum after intrauterine infusion of a low dose of prostaglandin F2α (PGF2A) aimed at enhancing our understanding of the mechanisms of luteolysis. Holstein dairy cows were superovulated (>6 corpora lutea [CL]) and on day 9 of the estrous cycle were infused with a low dose of PGF2A (0.5 mg PGF2A in 0.25 ml phosphate buffered saline) into the greater curvature of the uterine horn ipsilateral to the CL. Ultrasound-guided biopsy samples of different CL were collected at 0 min, 15 min, 30 min, 1h, 2h and 6h after PGF2A infusion. Expression profiles and localization of mRNA for PTGFR, CCL2/MCP-1, and immediate early genes (NR4A1, EGR1 and FOS), were investigated by using qPCR and in situ hybridization. The concentrations of early response genes including FOS, NR4A1, and EGR1 exhibited the greatest increase at 30 min after PGF2A, compared to other time points. Expression profile of CCL2 mRNA increased gradually after intrauterine infusion of PGF2A with maximal up-regulation for CCL2 at 6h. Abundance of PTGFR mRNA only increased at 15 min and significantly decreased at 6h, compared to 0 min. Cellular localizations of all studied genes except CCL2 (primarily localized to apparent immune cells) were predominantly visualized in large luteal cells. Interestingly, early response genes demonstrated a changing profile in cellular localization with initial responses appearing to be in both large luteal cells and endothelial cells, although no staining for PTGFR mRNA was observed in endothelial cells. Later, sustained responses, were only observed in large luteal cells, although PTGFR mRNA was decreasing in large luteal cells over time after PGF2A. The involvement of the immune system was also highlighted by the immediate increases in CCL2 mRNA that became much greater over time as there was an apparent influx of CCL2-positive immune cells. Thus, the temporal and cell-specific localization patterns for the studied mRNA demonstrate the complex pathways that are responsible for initiation of luteolysis in the bovine CL.