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Our objective was to determine the effects of a single treatment of human chorionic gonadotropin (hCG) or GnRH from d 5 to 7 of the estrous cycle on cycle length, expression of estrus and fertility in lactating dairy cows. Lactating Holstein cows (n = 354) located in Farm 1 and lactating Jersey cows located in Farm 2 (n = 210) detected in estrus by an Automated Activity Monitor (AAM) system from 27 to 50 days in milk (DIM) were randomly assigned to receive one of three treatments from d 5 to 7 of the estrous cycle: control (untreated; CON; Holstein, n = 111; Jersey, n = 66), GnRH (86 μg gonadorelin acetate im; Holstein, n = 116; Jersey, n = 75), or hCG (3,300 IU im; Holstein, n = 127; Jersey, n = 69). Ovaries were scanned with ultrasound in a random subgroup of cows (Holstein/Farm 1, n = 147; Jersey/Farm 2, n = 94) on the day of treatment and 3 or 4 d later to determine ovulation. Estrus was detected after treatment by an AAM, and peak activity and heat index were recorded. A random subgroup of cows observed in estrus after treatment received first AI from 51 to 80 DIM (Holstein, n = 208; Jersey, n = 138). Pregnancy diagnoses were performed by transrectal ultrasonography at 37 ± 3 d post-AI. Holstein and Jersey cows treated with GnRH and hCG had an increased (P 0.05) ovulatory response compared with controls. Human chorionic gonadotropin decreased (74%; P = 0.05) and GnRH tended to reduce (75%; P = 0.07) the proportion of multiparous Holstein cows returning to estrus compared with CON (86%). Cows treated with hCG had a longer (P 0.01) estrous cycle length (24.6 ± 0.3 d, Holstein; 23.0 ± 0.3 d, Jersey) compared with CON cows (22.7 ± 0.3 d, Holstein; 21.3 ± 0.3 d Jersey) and GnRH (22.9 ± 0.3 d, Holstein; 21.1 ± 0.3 d Jersey). The percentage of cows with high (≥80) peak activity and heat index did not differ (P 0.50) between treatments, and milk production did not affect (P 0.65) the duration of estrus. Pregnancy per AI (P/AI) was not affected by treatments in Holstein (P = 0.93; CON: 34.3%, GnRH: 35.4%, and hCG: 31.5%) and in multiparous Jersey cows (P = 0.35; CON: 34.3%, GnRH: 35.4%, and hCG: 31.5%), but hCG had greater (P = 0.03; 55%) P/AI than GnRH (30.0%) and a trend (P = 0.06) for greater P/AI than CON (33.3%) in primiparous Jersey cows. In summary, inducing the formation of an accessory corpus luteum from d 5 to 7 of the estrous cycle with hCG reduced expression of estrus in multiparous Holstein cows. Moreover, hCG increased estrous cycle length in Holstein and Jersey cows, and it did not affect first service P/AI at 37 ± 3 d post-AI in Holstein and multiparous Jersey lactating cows. However, hCG increased P/AI in primiparous Jersey cows. Future research with a larger number of cows is needed to confirm these intriguing fertility results.

Peroxiredoxins (Prdxs) are known to play a critical role in regulating male fertility as antioxidant enzymes. Although several studies have suggested a close association between Prdxs and male fertility, few studies have explored the efficacy of Prdxs to predict male fertility. Therefore, the current study was designed to discover the most efficient biomarkers among the Prdxs with six isoforms. Our study showed a significant positive correlation between the litter size and the levels of PRDX 4 among all isoforms in spermatozoa. Subsequently, a regression analysis using a combination of markers was conducted to increase efficacy for fertility prediction. Nevertheless, PRDX4 had the highest efficacy compared to other combination models to predict litter size. The prediction accuracy of male fertility was further evaluated through receiver operating characteristic curve analysis, which showed that PRDX 4 could predict the litter size with high overall accuracy of 95%. Moreover, litter size was increased by 1.55 piglets after predicting high litter size using PRDX 4. This is the first study to comprehensively elucidate the role of all isoforms of PRDXs on male fertility to the best of our knowledge. PRDX 4 was tested and evaluated up to a practical level. Data here reported suggesting PRDX 4 marker allowed the highest accuracy for male fertility prediction and diagnosis, leading to a measurable improvement in the male fertility outcome.

During an ovulatory follicular wave in the monovulatory species of heifers, mares, and women, the two largest follicles deviate in diameter at the end of a common follicle growth phase. The largest follicle before deviation becomes the future ovulatory follicle in most ovulatory waves. In 10-30% of the ovulatory waves, the destiny of the two follicles switches just before or at deviation so that the second-largest follicle becomes the future ovulatory follicle, and the largest follicle becomes a subordinate. In FSH-driven switching in heifers, mares, and women, the wave-stimulating FSH surge decreases to a low concentration before the largest follicle has developed the ability to utilize the low concentrations. The concentrations of FSH then increase (mares, women) or cease to decrease (heifers), and the next largest follicle acquires the capability of becoming the future ovulatory follicle. Luteolysis-driven switching has been reported in heifers but not in mares and women. The switching in heifers occurs during ovulatory wave 3 of three wave interovulatory intervals (IOI) when the wave of follicles is in the common growth phase in synchrony with the time of luteolysis. Regression of the CL during the common growth phase of ovulatory wave 3 is accompanied by decreased activity of follicles that are adjacent to the regressing CL but not when follicles and CL are separated or in opposite ovaries. The role of luteolysis in switching in heifers has been tested by treating with PGF2α when the largest follicle of wave 2 was near the end of the common growth phase. Switching in destiny of the largest follicle from the expected future dominant to a future subordinate occurred in most waves (10 of 17) when the largest follicle and regressing CL were in the same ovary and adjacent but not when separated in the same ovary or when in opposite ovaries (0 of 11). The newly selected future ovulatory follicle may develop in the opposite ovary. Thereby, frequency of the contralateral vs ipsilateral relationship between the preovulatory follicle and CL in heifers is greater in three-wave IOI than in two-wave IOI. In summary, the second largest predeviation follicle becomes the postdeviation dominant follicle when the decreasing FSH is out of phase with the largest predeviation follicle in heifers, mares, and women or when luteolysis and predeviation are in synchrony in heifers.

Notch signaling pathway plays an important regulatory role in the development of mammalian follicles. This study aimed to explore the effect of Notch2 on the function of bovine follicles luteinized granulosa cells (LGCs). We detected that the coding sequence (CDS) of bovine Notch2 gene is 7416 bp, encoding 2471 amino acids (AA). The homology of Notch2 AA sequence between bovine and other species is 86.04%-98.75%, indicating high conservatism. Immunohistochemistry found that Notch2 receptor and its ligand Jagged2 localize in granulosa cells (GCs) and theca cells in bovine antral follicles. And immunofluorescence found that positive signals of Notch2 and Jagged2 overlap in bovine LGCs, speculating that Notch2 receptor may react with Jagged2 ligand to activate Notch signaling pathway and play an important role in bovine LGCs. To further investigate the function of Notch2, Notch2 gene was silenced by short hairpin RNA (shRNA) and CCK-8 analysis showed that the proliferation rate of LGCs was downregulated significantly (P 0.01). Quantitative reverse transcription polymerase chain reaction (qRT-PCR) showed that the mRNA expression of apoptosis related gene Bcl-2/Bax decreased (P 0.01) and Caspase3 increased (P 0.05), cell cycle related gene CyclinD2/CDK4 complex decreased (P 0.01) and P21 increased (P 0.05), steroidogenesis gene STAR and 3β-HSD decreased (P 0.01) while CYP19A1 and CYP11A1 had no significant difference (P 0.05). In addition, Enzyme-linked immunosorbent assay (ELISA) showed that there was no difference in estradiol (E

This study was conducted to elucidate the effects of introducing conjugated linoleic acid (CLA) on meiotic spindle organization of heat stressed (HS) matured oocytes and the resulting blastocysts DNA methylation as well as the expression of the genes involved in DNA methylation (DNMT3a, DNMT3b and DNMT1). Immature bovine oocytes were cultured at 41 °C for the first 12 h and 38.5 °C for the second 12 h of maturation time in the presence of 0 and 50 μM of CLA (HS and HS + CLA groups, respectively). A group of oocytes cultured in medium with no CLA supplementation at normal temperature (38.5 °C for 24 h) was considered as negative control (C). Percentage of normal spindle, and cleavage and blastocyst rates were significantly decreased in the HS group compared to the C group (P 0.05). The global DNA methylation and expression level of DNMT3a gene were increased in HS group compared to the C groups (P 0.05), while the expression level of DNMT3b was decreased. The CLA supplementation improved the percentage of normal microtubules shape in MII oocytes as well as the developmental competence in the HS + CLA group compared to the HS group (P 0.05). However, global DNA methylation and expression level of DNMT3a/b were not ameliorated by CLA supplementation (P 0.05). Based on the obtained results, CLA proved to be capable of improving the oocyte developmental competence as well as decreased the aberrant spindle organization of heat-stressed oocytes and it would not cause epigenetic alteration in the obtained blastocysts.

Although prostaglandins are important in the ovulation process, a precise role for prostaglandin F2α (PGF) has not been elucidated. This study aimed to evaluate the regulation of PGF receptor mRNA (PTGFR) in granulosa cells and the local effect of PGF on ovulation and luteinization. In Experiment 1, using samples collected in vivo before (Day 2), during (Day 3) and after (Day 4) follicular deviation, expression of PTGFR in bovine granulosa cells was more abundant in the dominant follicle after deviation than in subordinates (P 0.05). However, the expression of PTGFR was not regulated (P = 0.1) in preovulatory follicles at different time-points (0, 3, 6, 12 and 24 h) after ovulation induction with GnRH. In Experiment 2, to assess the role of systemic PGF treatment on luteinization and vascularization of preovulatory follicles, flunixin meglumine (FM), a nonsteroidal anti-inflammatory drug, was used to inhibit endogenous prostaglandin synthesis. Cows with preovulatory follicles were induced to ovulate with GnRH (0 h) and allocated to three groups: Control, with no further treatment; FM, treated with 2.2 mg/kg FM im 17 h after GnRH treatment; and FM + PGF, treated with FM 17 h after GnRH, followed by 25 mg dinoprost tromethamine (PGF) 23 h after GnRH treatment. FM injection was able to reduce the concentration of PGF in the follicular fluid (FF) (P 0.001). However, contrary to our hypothesis, color Doppler ultrasound evaluations revealed decreased vascular flow in FM + PGF group (P 0.05), and no effect of the treatments on intrafollicular P4 and E2 concentrations 24 h after GnRH. The prostaglandin metabolite (PGFM) concentrations in the FF were greater in cows receiving systemic PGF (P 0.001), which prompted us to further check its role on ovulation. Therefore, in Experiment 3, in a final attempt to demonstrate the local effect of PGF on ovulation, cows with preovulatory follicles received an intrafollicular injection (IFI) of PBS (Control) or 100 ng/mL purified PGF (PGF group). PGF treatment did not affect the time of ovulation after IFI (66 ± 6.4 and 63 ± 8.5 h for control and PGF, respectively; P 0.05), further suggesting that it has no direct effect in the ovulatory process. Based on our findings, we concluded that FM decreased PGF synthesis within the follicle, whereas PGF treatment decreased follicular vascularization. In addition, the in vivo model of intrafollicular injection evidenced that PGF alone is not able to locally induce ovulation.

The aim of this study was to investigate the rapid response pathway and gene and protein expression profiles of the rat testis in response to estradiol (E2) and 1α,25(OH)2 vitamin D3 (1,25-D3), to understand how they mediate their effects on the first spermatogenic wave. To do this, we compared the effects of 1,25-D3 and E2 on 45calcium(Ca2+) uptake and the involvement of estrogen receptors (ESR) in their rapid responses. Additionally, we studied the downstream signal transduction effects of 1,25-D3 and E2 on cyclin A1/B1 and cellular cycle protein expression. As previously observed for 1,25-D3, E2 also increased 45Ca2+ uptake in immature rat testes via voltage-dependent Ca2+ channels, Ca2+-dependent chloride channels and via the activation of protein kinase C, protein kinase A and mitogen-activated protein kinase kinase (MEK). Elevated aromatase expression by testes was observed in the presence of 1,25-D3 and both hormones decreased ESR mRNA expression. Furthermore, 1,25-D3 and E2 diminished cyclin A1 mRNA expression, but E2 did not affect cyclin B1 mRNA levels. Consistent with these findings, the immunocontent of cyclin A1 and B1 in the testes was also increased by 1,25-D3 and E2. 1,25-D3 increased expressions of the p16 and p53 proteins, supporting the anti-proliferative and pro-apoptotic properties of 1,25-D3, while E2 also augmented p16. Data indicate that both hormones trigger rapid responses at the plasma membrane that may control the expression of gene and proteins related to cell cycle regulation, and thereby modulate spermatogenesis.

The use of worldwide glyphosate-based herbicide Roundup® is growing and to date its effects on mammalian spermatozoa are controversial. This study aims to investigate the functional impact of in vitro exposure of pig spermatozoa to low concentrations of Roundup® Ultra Plus (RUP), similar to those present as environment contaminants, to its active ingredient glyphosate, and to the non-active component, surfactant polyoxyethyleneamine (POEA). Pig spermatozoa were incubated in Tyrode's basal medium (TBM) or Tyrode's complete medium (TCM) (1 h at 38.5 °C) with several RUP dilutions or equivalent concentrations of glyphosate or POEA. RUP treatment causes a significant dilution-dependent decrease in sperm motility, a significant increase in plasma membrane disorganization and reduction in GSK3β phosphorylation (TBM) and in two PKA substrates (TBM and TCM), whereas does not affect sperm viability or mitochondrial membrane potential (MMP). Equivalent glyphosate concentrations do not affect any functional sperm parameters. However, POEA concentrations equivalent to RUP dilutions mimic all RUP sperm effects: decrease sperm motility in a concentration-dependent manner, increase sperm plasma membrane lipid disorder and significantly inhibit GSK3β phosphorylation (TBM) and two PKA substrates without affecting sperm viability or MMP. In summary, low concentrations RUP herbicide cause sperm motility impairment without affecting sperm viability. This adverse effect could be likely due to a detrimental effect in the plasma membrane lipid organization and to inhibition of phosphorylation of both, GSK3β and specific PKA substrates. Importantly, our results indicate that negative effects of low RUP concentrations in pig spermatozoa function are likely caused by the surfactant included in its formulation and no by its active ingredient glyphosate.

The aim of this study was to determine the cellular and molecular mechanisms of leptin (LEP) and the leptin receptor (LEPR) in testicular development of prepubertal ganders. In an in vivo animal experiment, active immunization against LEPR severely depressed prepubertal testicular development by significantly reducing testicular weights at 200 and 227 days of age. The number of elongated spermatids in the seminiferous tubules was also significantly decreased by immunization with LEPR at ages of 200 and 227 days. Inhibition of testicular development by LEPR immunization was associated with decreases in LHR, StAR, 3β-HSD, CYP11A1, CYP17A1, and PRLR mRNA expression levels in testicular tissue, which resulted in a significant decrease in testosterone synthesis. In the in vitro experiments, the addition of LEP combined with anti-LEPR antibodies strengthened LEPR signal transduction, and inhibited significantly testosterone production in cultured Leydig cells isolated from prepubertal gander testes. The mRNA expression of LHR, StAR, 3β-HSD, CYP11A1, CYP17A1 also decreased significantly after treatment with LEP combined with anti-LEPR antibodies in cultured Leydig cells. These results suggest that anti-LEPR antibodies strengthen LEPR signaling transduction in the presence of LEP, and immunization against LEPR inhibited testes development and testosterone secretion in prepubertal ganders.

Kisspeptins (KISSs) and RFamide-related peptide-3 (RFRP-3) affect the synthesis and secretion of luteinizing hormone (LH) and modulate female reproductive processes. The presence of KISS and RFRP-3 in the porcine pituitary gland and their contribution to the regulation of follicle-stimulating hormone (FSH) synthesis and secretion is unknown. This study analyzed the presence of KISS and RFRP-3 in the pituitary of estrous-cyclic pigs on days 2 to 3, 10 to 11, 12 to 13, 15 to 16 and 19 to 20 and early pregnant pigs on days 10 to 11, 12 to 13 and 15 to 16, and evaluated the effect of KISS and RFRP-3 on β-Fsh mRNA expression and FSH secretion in vitro by pituitary cells collected on selected days of the estrous cycle. The cells were cultured in vitro and treated with KISS (10−6 M, 10−7 M) and RFRP-3 (10−6 M, 10−7 M) or gonadotropin-releasing hormone (GnRH; 100 ng/mL) alone and in combinations (4 h or 24 h). The relative abundance of Kiss and Rfrp-3 and their receptor mRNA transcripts, as well as the KISS and RFRP-3 proteins, were found in the pituitaries of estrous-cyclic and early pregnant pigs. KISS after 4 h increased the secretion of FSH in estrous cyclic pigs mostly during the early-luteal phase and luteolysis. RFRP-3 inhibited the synthesis and secretion of FSH in estrous-cyclic pigs on days 19 to 20 and the secretion of FSH on days 2 to 3 and 10 to 12 of the estrous cycle compared with GnRH-treated cells. KISS in co-treatment with GnRH after 24 h enhanced FSH release on days 2 to 3 and 15 to 16 of the estrous cycle. In conclusion, KISS and RFRP-3 systems are present in the pituitary of estrous-cyclic and pregnant pigs. In estrous-cyclic pigs, KISS and RFRP-3 may affect the synthesis and secretion of FSH by pituitary cells.

Control of the farrowing process is crucial to reduce the risk of post-parturient disorders in sows and newborn piglet mortality. Carbetocin is a long-acting oxytocin-like compound that has been introduced to domestic animal obstetrics. The aims of the present study were to evaluate the effects of carbetocin administration during the mid-period of parturition on farrowing duration as well as colostrum and milk production and its side effects on neonatal piglet characteristics in hyperprolific sows. The study included 89 Landrace x Yorkshire crossbred sows and 1534 piglets. Sows were randomly distributed into two groups: CONTROL (n = 46), where sows were allowed to farrow naturally, and TREATMENT (n = 43), where sows were administered 0.5 μg/kg carbetocin intramuscularly after the birth of the 7th piglet. In each litter, the piglets were classified into two groups according to birth order: 1-7 and ≥8. The colostrum yield of sows and the colostrum intake of each individual piglet were calculated. Litter weight gain of the piglets from 3 to 5 days of lactation was used to estimate milk yield. Farrowing duration tended to be reduced from 338.4 ± 35.2 min in the CONTROL group to 255.3 ± 36.4 min in the TREATMENT group (P = 0.105). However, the colostrum yield in the TREATMENT group was lower than that in the CONTROL group (3828 ± 149 and 3041 ± 158 g, respectively, P 0.001). Nevertheless, milk yield during 3-5 days of lactation in the TREATMENT group did not differ significantly compared to that in the CONTROL group (7.53 ± 0.01 and 7.54 ± 0.01 kg/day, respectively, P = 0.643). The incidence of stillbirths in the TREATMENT group was not significantly different from that in the CONTROL group (8.3 and 7.4%, respectively, P = 0.944). However, the incidence of piglets born with umbilical cord rupture in the TREATMENT group with birth order ≥8 was higher than that in the CONTROL group in the same birth order (16.6 and 5.7%, P 0.001). Across groups, colostrum intake of piglets born with birth order ≥8 was lower than that of piglets born in the birth orders 1-7 (187.4 ± 8.7 and 241.5 ± 9.0 g, P 0.001). Interestingly, the colostrum intake of piglets with birth orders 1-7 in the TREATMENT group was also lower than that of piglets with the same birth order in the CONTROL group (214.9 ± 12.9 and 268.1 ± 12.5 g, respectively, P = 0.003). Colostrum intake was lowest in the piglets with birth order ≥8 in the TREATMENT group (159.6 ± 12.3, P 0.001). Carbetocin administration after the birth of the 7th piglet reduced the birth interval of piglets but increased the incidence of piglets born with umbilical rupture and reduced colostrum intake. Therefore, colostrum supplementation and care of the newborn piglets immediately after birth are strongly recommended after carbetocin administration.

As a promising strategy in overcoming drug resistance, the nano drug co-delivery system (NDCDS) can transport two or more drugs into the cell. In this study, we sought to compare the dual and single drug-delivery system, to deliver the optimal dose of Resveratrol (RES) and Tretinoin (TTN) into the in vitro matured oocyte and morula-compact stage embryonic cells. The formation of single (RES/TTN) and dual-drug (RES + TTN)-SLN were confirmed by Uv–vis spectrophotometery, dynamic light scattering (DLS), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) technologies. In two experiments, the oocytes/presumptive zygotes were cultured under various concentrations of the single (RES/TTN) and dual-drug (RES + TTN)-SLN. In vitro toxicity studies, including nuclear staining (Aceto-orcein and Hoechst 33342), H2DCFDA fluorescent staining, chemiluminescence assay, and quantitative reverse transcription-PCR (qRT-PCR) techniques, indicated an excellent oocyte/embryo internalization of RES and TTN. Moreover, when oocytes/embryos were treated with the lowest concentration of RES + TTN-SLN, antioxidants-related genes were upregulated, apoptotic-related genes were downregulated, and intra/extracellular ROS production was reduced. In vitro cytotoxicity studies also demonstrated that single/dual-encapsulation of RES or TTN were safe even at the highest concentration (10 and 5 μM) compared to the control group. To sum it up, both delivery systems of RES and TTN by SLN (dual or single encapsulation) can deliver the optimal dose of RES and TTN into the oocyte/embryo. Where the dual-delivery of RES and TTN even at the lowest concentration (0.25 μM + 0.1 μm) showed a synergistic anti-oxidative effect in oocyte/embryo with a better inhibition of intra/extra-cellular ROS production by an enhanced/controlled intracellular penetration.

The objective of this study was to evaluate the effect of exposure to different colors of light during egg incubation on the reproductive parameters of male and female Japanese quails. A total of 1776 eggs were incubated under four lighting conditions for 24 h a day during the entire incubation period: white LEDs, green LEDs, red LEDs and darkness (control). The experimental design was a randomized block (incubation time) with four treatments of six replicates of two cages each. After hatching, the birds were housed in brood cages with 18 birds each to 35 days of age, when they were sexed and transferred to production cages for evaluation of reproductive parameters. After the onset of laying, the number of eggs in each cage was recorded daily, and the values were used to obtain the age of the females at first egg and at 80% laying. At 35 and 60 days of age, several birds from each cage were euthanized for anatomical and histological evaluation of the gonads. Two females from each cage were weighed every three days until 60 days of age to determine the growth curve. After 60 days, eggs from each cage were collected and assessed for external and internal quality. At 70, 74 and 78 days of age, semen collection was performed and seminal quality was evaluated. Then, the males were transferred to cages containing 9 females for the fertility test. Hatchability was higher (P 0.05) in eggs incubated in the dark and under the red LED. The age of maximum growth was higher (P 0.05) in birds from eggs incubated in the dark and under the white LED. There was no difference (P 0.05) in the anatomical and histological characteristics of the testicles between the groups incubated under different light colors, except for the diameter of the seminiferous tubules, which was greater (P = 0.05) in the dark and in the white LED groups. There was no effect (P 0.05) of light color during incubation on the productive index or egg quality of adult birds. There was also no effect (P 0.05) on sperm quality, except for sperm motility, the values of which were higher (P 0.05) in birds from eggs incubated in different colors of light. However, this difference was not sufficient to significantly (P 0.05) influence bird fertility. It is concluded that under the studied conditions, the incubation of quail eggs under white, red, and green LED lamps does not influence the reproductive characteristics of the quails.

The study describes the details of histological changes occurring during the postnatal development of the wall of the uterine horn in the European shorthair domestic cat. Light microscope observations and morphometric analysis were carried out to investigate the rate of development of the wall of the uterine horns in order to estimate the maturing time. Uterine horns of newborn and 1week-old cats are fetal-like and showing smooth, nonfolded endometrium lined with pseudostratified epithelium, deprived of uterine glands, and poorly developed two-layered myometrium. The lamina propria of the mucosa becomes folded in the first month of postnatal life. At the same time, the primordia of the uterine glands appear, elongating, doubling in number and reaching the basal layer by sixth month of age. In six-month-old cats, the lamina propria of the mucosa is formed into the functional and basal layer and its thickness increases rapidly. Until the sixth month of age, the inner circular muscle layer is the widest part of the myometrium, the outer longitudinal layer is the thinnest, while the width of the vascular layer is an intermediate value between the width of the inner and outer layer. From the seventh month of age, the proportion of the thickness of the inner and outer layer is reversed and the vascular layer becomes the thinnest part of the myometrium. Between seventh and twelfth month of age, the structure of the wall of the uterine horn does not change significantly, as the longitudinal mucosal folds limiting the narrow lumen of the uterine horns, and the arrangement of the layers in the wall is assigned to a pattern according to which the endometrium represents about 40% of the total width of the wall, while the myometrium is about 60%. In the studied time span of postnatal life, the average thickness of the wall of the uterine horn increases 9.4 -fold, the thickness of the lamina propria and mucosal folds increases 6.9-fold and 3.8-fold, respectively, while the thickness of the myometrium increases 17.3-fold. Summing up, the process of maturation of the wall of the uterine horns in the domestic cat lasts up to sixth month of postnatal life and includes the formation of the endometrial longitudinal folds, appearance and development of the uterine glands, division of the lamina propria of the mucosa into the functional and basal layers, and continued development of the circular, vascular and longitudinal muscle layers.

The main objectives of this study was to identify the effects of a relationship of hyper-concentration of Gamma-glutamyltransferase (γ-GTP) in follicle fluid (FF) on the levels of glutathione (GSH)/reactive oxygen species (ROS) in oocytes and subsequent embryo development in cattle with abnormal livers. Furthermore, we investigated the effect of supplementing in vitro maturation medium with glutathione ethyl ester (GSH-OEt) on the subsequent developmental potential of oocytes from such cattle. We used a control group of cattle (with normal livers) and a liver disorder (LD) group, in which the liver was diagnosed as being abnormal. In experiment 1, the LD group was divided to two subgroups according to the concentration of γ-GTP in FF: a low group (≤50 IU/L; the low LD group), and a high group (>50 IU/L: the high LD group). Cumulus oocyte-complexes (COCs) were matured and fertilized in vitro and then cultured to the blastocyst stage. The levels of GSH and ROS in the matured oocytes after IVM were then assessed in each group. On day 7 after fertilization, embryo cleavage and development were assessed. We found that the rate of development to the blastocyst stage was significantly lower in the high LD group than in the control group and the low LD group. The levels of GSH in matured oocytes were significantly lower in the high LD group than in the control group and low LD group. The levels of ROS in matured oocytes was significantly higher in the high LD group than in the control group and the low LD group. In experiment 2, COCs from cattle in the high LD group were matured in m-199 supplemented with 5 mM GSH-OEt, then IVF and IVC was performed for 7 days. The GSH levels were determined in some COCs after IVM. The supplementation of media with GSH-OEt during IVM increased the levels of GSH in mature oocytes and improved the rate of blastocyst development compared with the control group. In conclusion, GSH-OEt supplementation to media during IVM can improve the developmental potential of oocytes in liver-diseased cattle with high γ-GTP concentrations in the FF by increasing intracellular GSH synthesis and scavenging ROS.

In batch spawning fish, secondary growth oocytes (SGO) are recruited and spawned in successive cohorts, and multiple cohorts co-occur in spawning-capable females. So far, histological features such as the prevalence of cortical alveoli or yolk granules are conservatively used to distinguish oocytes in different developmental stages which do not necessarily correspond to different cohorts. In this way, valuable information about spawning dynamics remains unseen and consequently misleading conclusions might be drawn, especially for species with high spawning rates and increased overlapping among oocyte cohorts. We introduce a new method for grouping oocytes into different cohorts based on the application of the K-means clustering algorithm on the characteristics of cytoplasmic structures, such as the varying size and intensity of cortical alveoli and yolk granules in oocytes of different development. The method allowed the grouping of oocytes without the need of using oocyte diameter, and thus, a crucial histological bias dealing with the cutting angle and the orientation of reference points (e.g. nucleus) has been overcome. Using sardine, Sardina pilchardus, as a case study, the separation of cohorts provided new insight into the ovarian dynamics, indentifying successive recruitment of up to five oocyte cohorts between SGO recruitment and spawning. These results verified previous histological indications of the number of cohorts in sardine. Altogether, this method represents an improved tool to study species with complex ovarian dynamics.

The present study was carried out to compare the proteomic profiles of spermatogenic cells of crossbred and zebu cattle in an effort to understand the possible reasons for a higher incidence of sub-fertility in crossbred bulls. The spermatogenic cells collected from the testes of pre-pubertal (6 mo) and adult (24 mo) crossbred and zebu males through fine needle aspiration were proliferated in vitro, and proteomic profiling was done using a shotgun proteomics approach. The age- and species-specific variations in the expression level of proteins were identified in spermatogenic cells. The number of differentially expressed proteins (DEPs) identified in pre-pubertal zebu and crossbred was 546, while 579 DEPs were identified between adult zebu and crossbred bulls. Out of these, 194 DEPS were common to these groups and 40 DEPs displayed a fold change ≥2. However, only 20 proteins exhibited similar expression variation trends (upregulated or downregulated) among pre-pubertal as well as adult zebu and crossbred bulls. Out of these 20 DEPs, 13 proteins were upregulated, and 7 proteins were downregulated in spermatogenic cells of zebu compared to crossbred bulls. Among the upregulated proteins were RPLP2, PAXIP1, calumenin, prosaposin, GTF2F1, TMP2, ubiquitin conjugation factor E4A, COL1A2, vimentin, protein FAM13A, peripherin, GFPT2, and GRP78. Seven proteins that were downregulated in zebu bulls compared to crossbred included APOA1, G patch domain-containing protein 1, NAD P transhydrogenase mitochondrial, glutamyl aminopeptidase, synaptojanin 1 fragment, Arf GAP with SH3 domain ANK repeat and PH domain-containing protein 1, and protein transport protein sec16B. It was inferred that the proteins associated with sperm function and fertilization processes, such as calumenin, prosaposin, vimentin, GRP78, and APOA1 could be studied further to understand the precise cause of subfertility in crossbred bulls.

Commercially available intravaginal progesterone (P4) devices differ in shape, surface area and P4 load, which may affect the resulting pregnancy per AI (P/AI) following timed-AI (TAI). The objective of this study was to compare two intravaginal P4 devices on estrus rate, follicular dynamics and P/AI in beef cattle subjected to shortened-TAI protocols. In Expt. 1, nulliparous heifers were randomly assigned to a P4-releasing intravaginal device (PRID-Delta, 1.55 g P4) or a controlled internal drug release (CIDR, 1.38 g P4) at the initiation of a J-synch protocol. Heifers that displayed estrus 72 h following device removal were TAI, or if not in estrus given GnRH at 72 h and TAI at 90 h. In Expt. 2, nulliparous heifers and non-suckling cows were randomly assigned to either PRID or CIDR groups and either 1 or 2 mg of estradiol benzoate (EB) at initiation of a J-synch protocol. All cattle were TAI concurrent with GnRH 72 h after device removal. In Expt. 3, nulliparous heifers and suckling cows were randomly assigned to either PRID or CIDR groups and initiated a 5-d Cosynch protocol, with TAI concurrent with GnRH 72 h following device removal. In each experiment, cattle received estrus detection patches at device removal, which were then scored from 0 to 3 based on color change between initial application and TAI; 0 = unchanged, 1 = ≤50% change, 2 = >50% change, 3 = missing. Estrus was defined to have occurred when the patch was scored 2 or 3. Transrectal ultrasonography was used to determine cyclicity, diagnose pregnancy in all experiments, and the size of the ovulatory follicle in Expt. 3. In Expt. 1, the estrus rate was greater (72.0% vs. 61.0%; P = 0.04) in the PRID compared to the CIDR group. In Expt. 2, a parity by EB dose interaction (P = 0.02) was attributed to an increased estrus rate (52.8% vs. 41.4%; P = 0.05) in heifers given 1 vs. 2 mg EB. In Expt. 3, there was no difference in the ovulatory follicle diameter at device removal (P = 0.22) or TAI (P = 0.28) between P4 groups. Treatment with a PRID tended (P = 0.10) to increase the P/AI in cows compared to a CIDR (73.5% vs. 61.0%). In all experiments combined, the overall P/AI tended to increase (55.2% vs. 51.0%; P = 0.08) and P/AI in cattle exhibiting estrus increased (64.4% vs. 59.7%; P = 0.02) in cattle given a PRID compared to those given a CIDR, respectively. In summary, the type of intravaginal P4 device affected estrus response and P/AI following TAI in beef cows.

This study was performed to investigate the difference in developmental competence of oocytes derived from ovum pick-up (OPU) and slaughterhouse ovaries (SLH), and its underlying mechanisms. The OPU and SLH oocytes were in-vitro maturated and fertilized to produce blastocysts, and these blastoycsts were collected to explore the expression of key genes for developmental potential and telomere (Oct-4, Sox2, Nanog, Cdx2, Gata3, E-cadherin, β-catenin, TERT, TERF1 and TERF2). The results showed that both the cleavage and blastocyst rates were significantly higher for the OPU group (68.31%, 39.48%, respectively) than SLH group (57.59%, 26.50%, respectively) (P 0.01). The relative mRNA abundances of Sox2, Oct-4, Nanog and E-cadherin were significantly higher in the OPU blastocysts than the SLH ones (P 0.01). Protein expression analysis by Western blot and immunofluorescence also revealed that the expression of E-cadherin and Sox2 was significantly higher in OPU blastocysts than SLH ones. However, there was no significant differences between the two groups in the expression of Cdx2, β-catenin, Gata3, TERT, TERF1, TERF2. These results imply oocyte sources modify the expression of development and adhesion related genes in blastocysts, which may elucidate a possible reasoning for the low development competence of buffalo SLH embryos.

Today, orthodox medicine has almost exceeded its limits in resolving subfertility problems in animals, thus making phytomedicine a primary tool in the treatment of infertility. In this work, three herbal supplements obtained from freshly air-dried Moringa oleifera, Phyllanthus amarus and Viscum album leaves were evaluated to ascertain their comparative effect on the reproductive potentials of bucks. Sixty bucks were allotted four diets made up of standard grower rabbit ration without supplement and with 5% Moringa, Mistletoe and Phyllanthus supplementation for 84 days. Semen samples were collected from all bucks using artificial vagina, for semen quality and seminal oxidative stress markers. The organ weights, testicular and epididymal spermatozoa reserves were assessed to determine sperm production potentials using standard procedures. The result obtained revealed that spermatozoa concentration, progressive motility, curvilinear velocity, average path velocity and the amplitude of lateral head was significantly (p 0.05) higher in the group fed with mistletoe supplemented diet compared to that recorded in the control groups. The inclusion of herbal supplements linearly (p 0.05) increased the seminal total antioxidant activity with a corresponding decrease in the seminal lipid peroxidation across the herbal supplemented treatments compared to the control. The gonadal and extra-gonadal sperm reserves of bucks fed on the herbal supplements were depleted compared to bucks on control. Bucks on mistletoe supplementation rivalled the superior daily sperm production and testicular sperm reserve in bucks without herbal supplements. In conclusion, Viscum album supplements in bucks' diets encouraged daily sperm production, testicular sperm reserves, testosterone, as well as spermatozoa kinetics. The three herbal supplements did enhance semen oxidative stability.

During capacitation, proteins in boar sperm are released to maintain the stability of their own state and membrane structure. No studies have analyzed the differences between retained proteins and released proteins during sperm capacitation. In the present study, a Transwell chamber and polycarbonate membrane were used to separate the proteins of boar sperm and their released proteins. Isotopically labeled relative and absolute quantification (iTRAQ) was used to analyze each compartment protein. A total of 108 differential proteins were identified in the upper and lower chambers of the Transwell, among which 27 were significantly upregulated (p-value≤0.05 and |log2 (fold change)|≥1) and 81 were significantly downregulated (p-value≤0.05 and |log2 (fold change)|≤1). These differential proteins were mainly involved in biological processes (e.g., the regulation of cysteine peptidase activity, transmembrane transportation, ion transportation and ATP synthesis) and major signaling pathways (e.g., glutathione/galactose metabolism, cellular adhesion and PI3K-Akt), and most of them interacted with each other to some extent. In conclusion, retained proteins and released proteins of capacitated sperm were effectively separated using a Transwell chamber, and differential proteins were successfully identified from among the proteins. Bioinformatics analysis suggested that these differential proteins affect sperm capacitation mainly by adjusting sperm energy metabolism, motion characteristics and acrosome membrane status.

The objective of this study was to characterize circulating Anti-Müllerian Hormone (AMH) concentrations in a population of Holstein heifers and examine the impact that life events and stage of life have on those concentrations. Virgin, Holstein heifers (n = 105) 13 ± 0.8 months old were heat detected using tail-chalk, bred via artificial insemination and pregnancy checked 32+ days later. Serum samples for AMH were collected upon enrollment (heifer), at 5-20 days in milk (fresh) and at 45-60 days in milk (pre-breeding). Transrectal ultrasonography was performed upon enrollment (heifer) and at 45-60 days in milk (pre-breeding) to determine antral follicle count (AFC), cyclicity status, and uterine health. Heifers were blocked into thirds by AMH concentration: HIGH (354 pg/mL; n = 34), MID (183-354 pg/mL; n = 35) and LOW (183 pg/mL; n = 36), with distribution re-evaluated at subsequent samplings (fresh, pre-breeding). As heifers, age and conception risk to first service were not impacted by AMH (P 0.05). Reason for leaving the herd, health incidences and calving difficulty were not impacted by AMH (P 0.05). AFC and cyclicity had a positive impact on heifer AMH (P 0.01). AFC and AMH in heifers were highly correlated (0.56, P 0.001). AFC for heifers differed by AMH group with the HIGH group having the greatest AFC (8.76), followed by the MID (5.87), then the LOW (3.53) group (P 0.0001). However, this association was not evident in the pre-breeding group (P 0.05). From the heifer to the fresh sample, average AMH dropped from 313.15 pg/mL to 160.01 pg/mL (P 0.0001). Average AMH at the pre-breeding sample was 183.23 pg/mL, which was lower than the heifer sample (P 0.0001), but not different from the fresh sample (P 0.05). AFC and AMH at the heifer sample had a positive impact on AMH at the fresh sample (P 0.01) and pre-breeding AMH was positively impacted by both the fresh and heifer AMH concentration (P 0.001). Most animals kept their AMH categorization through all three time points with more of the LOW AMH animals maintaining their categorization than the other groups. However, 32.1% of animals changed their AMH categorization from the heifer sample to the fresh sample, with 53.8% moving to a lower AMH categorization (corresponding to lower AMH) and 46.2% moving to a higher AMH categorization (corresponding to higher AMH). No differences were seen in circulating AMH based on health events however, differences in AMH concentration over time indicate a drop in circulating AMH post-calving. Circulating AMH concentration as a pre-breeding heifer is highly indicative of circulating AMH concentration as a first lactation animal and may be used to predict an adult animal's AMH concentration. However, it is necessary to compare AMH concentrations to herdmates as published AMH values vary widely from herd to herd. In addition, sampling time should be considered when determining AMH categorization of animals as circulating AMH concentration immediately post-calving may not be indicative of an animal's true AMH categorization.

As one of the most powerful natural antioxidants, astaxanthin (Ax) has begun to be applied to the field of reproductive biology. Here we used porcine oocyte as a model to explore how Ax improves the oocyte potential during in vitro maturation (IVM), and we also investigated the cytoprotective effects of Ax on the vitrified oocytes. Ax supplementation (final concentration of 2.5 μM) was subjected for immature oocytes during vitrification and subsequent IVM; fresh oocytes were also matured in vitro in the presence or absence of 2.5 μM Ax. Our results showed that Ax significantly increased the survival rate of vitrified oocytes, and promoted the blastocyst yield of both fresh and vitrified oocytes after parthenogenetic activation and somatic cell nuclear transfer. The oocytes treated with Ax displayed significantly lower reactive oxygen species generation and higher glutathione level. Vitrification of oocytes had no impact on caspase-3, cathepsin B and autophagic activities; Ax significantly decreased the cathepsin B activity in both fresh and vitrified oocytes. Moreover, the relative fluorescence intensity of lysosomes was significantly increased in vitrified oocytes, which was recovered by Ax treatment. The mitochondrial activity did not differ between fresh and vitrified oocytes, and was significantly enhanced in Ax-treated oocytes. Furthermore, Ax significantly restored the decreased expression of BMP15, ZAR1, POU5F1, GPX4 and LAMP2 genes in vitrified oocytes. Both fresh and vitrified oocytes treated with Ax showed significantly higher mRNA levels of GDF9, POU5F1, SOD2, NRF2 and ATG5. Taken together, this study provides new perspectives in understanding the mechanisms by which Ax improves the developmental competence of both fresh and vitrified porcine oocytes.

The aim of this study was to evaluate the outcomes of an early resynchronization protocol (Resynch) initiated at different timepoints after timed artificial insemination (TAI) and with unknown pregnancy status. Holstein cows (n = 164) were submitted to the following TAI protocol: D0, insertion of an intravaginal progesterone (P4) device and 2 mg im estradiol benzoate (EB); D8, removal of P4 device and treatment with 0.5 mg im sodium cloprostenol (PGF); D9, 0.1 mg im Lecirelin (LEC); and D10, TAI1. Cows were then randomly assigned to Resynch protocols starting either on day 20 (Resynch20D, n = 82) or 25 after TAI1 (Resynch25D, n = 82) with the insertion of a new P4 device and EB treatment. In both groups, P4 device was removed on day 8 after the beginning of Resynch, the same day of pregnancy diagnosis by ultrasonography. In pregnant cows there was no further action. Non-pregnant cows were treated with 0.5 mg im PGF, had a blood sample collected for serum P4 analysis and we measured and recorded the size of the largest follicle and the presence or absence of a corpus luteum (CL). One day later, cows were treated with 0.1 mg im LEC and TAI2 occurred 12-14 h later. The diameter of the largest follicle and serum P4 were compared between groups by ANOVA for the main effects of treatment, presence of a CL, and their interaction, whereas pregnancy per artificial insemination (P/AI) and the percentage of cows with a CL on the day of ultrasonography were analyzed using the Chi-square test. Follicle diameter on day 8 of Resynch was greater for cows in the Resynch20D group compared with Resynch25D (15.9 ± 3.9 vs 12.2 ± 2.5 mm, respectively; P = 0.046). The Resynch25D group had a greater percentage of cows with a CL (51.9 vs 18.9%, respectively; P = 0.0008) and higher serum P4 (2.8 ± 1.1 vs 1.7 ± 0.8 ng/mL; P = 0.041) at the end of the protocol compared with Resynch20D. P/AI at TAI1 was 35.4 and 36.6% (P 0.10) for cows enrolled in Resynch20D and Resynch25D groups, respectively. P/AI to TAI2, after Resynch protocols, was greater in Resynch25D than Resynch20D (44.2 vs 22.6%, respectively; P 0.05). In conclusion, starting an early resynchronization protocol 25 days after TAI increases P/AI compared with starting 20 days after TAI, and this was associated with a presumed greater proportion of cows with a functional CL at the moment of P4 device removal.

Although it is well established that chlamydial disease renders female koalas infertile, there has been limited research on its effects on male koala fertility, specifically sperm quality. This study determined whether chlamydial infection adversely affects semen quality of naturally infected koalas and spermatozoa recovered from Chlamydia negative koalas co-incubated in vitro with C. pecorum elementary bodies (EBs). Semen from 102 south-east Queensland sexually mature wild koalas exhibiting varying degrees of chlamydiosis and clinical signs of disease were assessed for semen quality and compared to 11 clinically healthy, Chlamydia-free captive male koalas. For in vitro studies, semen samples were collected from 6 Chlamydia-free captive koalas, and co-incubated over 24 h with high and low concentrations of C. pecorum EBs and sperm quality assessed. Wild koalas displaying severe signs of clinical disease with C. pecorum present in the semen had significantly greater sperm DNA damage (P = 0.0267). The total % of morphologically abnormal spermatozoa was highest in wild koalas that had severe signs of clinical disease but whose semen was negative for C. pecorum (P = 0.0328). This apparent contradiction is possibly associated with wild males having resolved the infection but still possessing underlining reproductive pathology. A higher incidence of loose head spermatozoa occurred in semen of wild koalas not infected with C. pecorum compared to those that were C. pecorum infected (P = 0.026). In vitro incubation of semen with C. pecorum significantly decreased sperm motility and viability over 24 h.

Early embryo development, implantation and pregnancy involve a complex dialogue between the embryo and mother. In cattle this dialogue starts as early as days 3-4 when the embryo is still in the oviduct, and it continues to implantation. Immunological processes involving cytokines, mast cells and macrophages form an important part of this dialogue. Amongst the cytokines, interleukin-6 (Il-6) and leukemia inhibitory factor (LIF) are secreted by both the embryo and uterine endometrium and form part of an ongoing and reciprocating dialogue. Mast cells and macrophages populate the uterine endometrium during embryo development and are involved in achieving the correct balance between inflammatory and anti-inflammatory reactions at the uterus that are associated with embryo attachment and implantation. Embryo loss is the major cause of reproductive wastage in cattle, and livestock generally. A deeper understanding of immunological processes during early embryo development will help to achieve the next step change in the efficiency of natural and assisted breeding.

The aberrant redox regulation and anti-oxidative defense is one of the main causes of age-induced decline in oocytes quality and embryo development in mammals. The present study aimed to elucidate the effect of glutathione ethyl ester (GSH-OEt), a cell-permeable glutathione (GSH) donor, on the developmental competence of oocytes in cows with advanced reproductive age. Oocytes were collected from cows aged 30–50 months or >120 months, which were defined as young or aged, respectively, and subjected to in vitro maturation (IVM) in the presence of 5 mM of GSH-OEt. In aged cows, the GSH level in follicular fluid was lower, and the intracellular levels of reactive oxygen species (ROS) in post-IVM oocytes was higher than those in young cows. GSH-OEt supplementation during IVM reduced the ROS contents of oocyte in aged cows but not in young cows. GSH-OEt treatment promoted the meiotic progression and increased the proportion of oocytes with mature cytoplasm containing evenly dispersed cortical granules in aged cows. After in vitro fertilization, the normal fertilization and development to the blastocyst stage were enhanced by GSH-OEt in aged cows to levels comparable to those in young cows. Further, oocyte maturation in the presence of GSH-OEt increased the proportion of diploid blastocyst in aged cows. In contrast, GSH-OEt failed to enhance the oocyte maturation, fertilization, and embryo development in young cows. Taken together, the exogenous supplementation of GSH-OEt during IVM modulated the age-related oxidative damage of bovine oocytes and improved the developmental competence of oocytes in aged cows. Oocytes presented a distinct response to GSH-OEt treatment depending on the donor age. GSH-OEt supplementation during IVM could be of practical value through the efficiency improvement of chromosomally normal embryo production in aged cows.

Sebastes schlegelii is a typical viviparous teleost with six months sperm storage duration from November to April. In this study, spermatozoa morphological and physiological characteristics and sperm location in the female ovary were investigated by electron microscopy, computer-assisted sperm analyzer and histologic analysis, respectively. During copulation, we observed that spermatozoa in the testis had mature structure with rod-shaped head, a short midpiece, and a long flagellum. And further verified sperm swam freely at a high speed in the ovary fluid. After copulation, we only found swimming sperm in the ovary fluid at the early storage stage (November to December) and the majority of sperm were scattered randomly in the ovary cavity and partially concentrated in the crypt between the oocyte and stalk of follicle. Thereafter, the ovarian epithelium around the oocytes proliferated rapidly and wrapping spermatozoa outside of the follicular layer and formed a lot of crypts outside of the follicular layer which served as the sperm storage site until fertilization. The present findings would be useful for further understanding the mechanism of long-term sperm storage in viviparous teleost.

Small ubiquitin-like modifier 2 (SUMO2) is a small protein that modulates the stability and activity of other proteins. Although a variety of activities have been attributed to SUMO2, its function in preimplantation embryos is still obscure. We first explored the expression of SUMO2 protein in early embryos, and showed that compared with the 2-cell stage, the expression was increased at first, peaked at the 8-cell stage, and then dramatically decreased. To study the function of SUMO2, we used siRNA microinjection to knock down SUMO2.The silencing of SUMO2 significantly reduced the rate of in vitro blastocyst development from 75.56% to 40.60%. Notably, knockdown of SUMO2 (KD) altered the expression of CDX2, OCT4, and NANOG. The number of cells expressing CDX2 decreased, while OCT4 and NANOG were ectopically expressed in siSUMO2 embryos. The global H3K27me3 levels in SUMO2-KD embryos also were lower than in untreated embryos. Taken together, SUMO2 appears to play a significant role in mouse preimplantation embryos probably through key epigenetic modifications and regulation of pluripotency genes.

The maturation kinetics and in vitro fertilization of immature bovine oocytes injected by the intra-follicular oocyte injection (IFOT) technique into pre-ovulatory follicles of previously synchronized cows were evaluated. In Experiment 1, grade I, II and III cumulus-oocyte complexes (COCs) were randomly distributed to one of three Groups: Matvitro22 (COCs matured in vitro for 22 h), MatFol20 and MatFol28 (COCs matured in vivo after being injected into a pre-ovulatory follicle of previously synchronized cows for 19.8 ± 0.1 h and 28.3 ± 0.1 h, respectively). Cows received 12.5 mg of LH (Lutropin, Bioniche, Canada) at the time of IFOT in the MatFol20 Group or 10 h after IFOT in the MatFol28 Group. MatFol20 and MatFol28 COCs were aspirated approximately 20 h after the LH injection for nuclear maturation kinetics and recovery rate assessment. In Experiment 2, grade I, II, and III COCs were randomly distributed into two Groups: Matvitro22 Group, COCs were matured and fertilized in vitro, and MatFol20 Group, COCs were matured as in the MatFol20 Group in Experiment 1, but COCs were fertilized in vitro. Putative zygotes were classified as fertilized, unfertilized or polyspermic. In Experiment 1, the recovery rate was lower (P 0.001) in the MatFol20 Group (52.9%, 91/172) compared with MatFol28 (72.9%, 113/155). Rate of oocytes in germinal vesicle stage, metaphase I, anaphase I and telophase I were similar among Groups. However, oocytes matured in vivo for 28.3 h had lower rate of metaphase II (P = 0.001) and greater rates of degenerated (P = 0.001) and parthenogenetically activated (P = 0.001) oocytes. In experiment 2, the rates of polyspermy and degenerated were similar between Groups. However, the rate of fertilized oocytes was greater (P = 0.05) in oocytes in the MatFol20 Group. It is concluded that oocyte in vivo maturation for 19.8 h after IFOT does not compromise the nuclear maturation kinetics and increases in vitro fertilization rates. However, the extra 10 h of intra-follicular incubation time decreased oocyte viability.

Mithun (Bos frontalis) is a unique domestic free-range bovine species available in North Eastern hilly (NEH) regions of India and is reared for its protein rich meat. Mithun suffers severe non-cyclical population fluctuations; however, it is not yet declared as endangered species. Mithun follows some sort of seasonality based on the calving trend and semen production, although it is a perennial breeder. However, there is need to study the rhythmic changes of endocrine profiles to understand the hormone flow pattern in mithun to select the suitable time for blood collection to assess the exact endocrine profiles and to select the suitable time for natural breeding or semen collection by artificial methods for further research, conservation and propagation of its germplasm. Therefore, the present study was designed to evaluate the reproductive as well as metabolic endocrinological profiles in 24:00 h in intact adult mithun bulls during different seasons (winter, spring, summer and autumn) to know the rhythmic changes and flow pattern of the endocrinological profiles to improve its reproductive efficiency. Experimental mithun bulls (n = 6; age: 5-6 years; body condition score: 5-6 out of 10, classified as good) were selected for the study. Endocrinological profiles, follicle stimulating hormone (FSH), luteinizing hormone/interstitial cell stimulating hormone (LH/ICSH), testosterone, cortisol, thyroxine (T4), insulin like growth factor-1 (IGF-1), prolactin and melatonin (MT) were estimated at 04:00 h interval for one whole day in four seasons. The analysis was completed in two different ways as different times of collection (08:00, 12:00, 16:00, 20:00, 24:00 and 04:00 h) and day (08:00 to 16:00 h)night time (20:00 to 04:00 h) collections. Repeated measures ANOVA analysis revealed that the bulls in winter and spring had significantly (p 0.05) higher FSH, LH, testosterone, T4, IGF-1 and MT than those in summer whereas the bulls in summer had significantly higher cortisol and prolactin than those in winter and spring seasons. Similarly FSH, LH, testosterone, T4, IGF-1 and MT were significantly (p 0.05) higher in night than in day time collections whereas cortisol and prolactin were significantly (p 0.05) higher in day than in night time collections in different seasons. Correlation analysis revealed that FSH, LH, testosterone, T4, IGF-1 and MT had significant (p 0.05) positive correlation with each other whereas these had significant (p 0.05) negative correlation with cortisol and prolactin. The study concludes that season and time of blood collection had significant effect on the endocrinological profiles in mithun bulls. Estimation of FSH, LH, testosterone, T4, IGF-1 and MT during night time and cortisol and prolactin during day time was more appropriate to get correct value of the endocrinological profiles. Spring and winter have significantly greater beneficial effects than summer on reproduction and artificial breeding programs in mithun species in the semi-intensive management in the present location.

Interspecific germline chimerism mediated by transplantation of primordial germ cells (PGCs) of wild species to domestic hosts promises the conservation of wild birds. Cryopreservation of avian eggs and embryos is impracticable, and currently only frozen PGCs enable conservation of both the male and female descendants. Purebred offspring have been obtained from germline chimeras of wild avian species, proving the feasibility of such technology. In vitro propagation has been optimized for avian PGCs of domestic species; however, evidence is rather limited for successful isolation as well as long-term culture from a single embryo of wild species. With accelerating biodiversity loss, we have committed to preserving current genetic resources by freezing PGCs isolated from individual embryos in addition to their genetic material. We have devised a reliable protocol for the isolation and proliferation of PGCs from wild fowls in the family Phasianidae that are conserved in captive breeding (red junglefowl, bar-tailed pheasant, kalij pheasant, Siamese fireback pheasant, and silver pheasant). We obtained individual isolates of cultured circulating PGCs (49.7%, 79/155) as well as tissue PGCs (92.9%, 144/155). The specific co-culture conditions of autologous embryonic cells, without additional growth factors, facilitated the proliferation of so-called tissue PGCs (the remaining PGCs in embryonic tissue following blood aspiration). Only circulating PGCs left in blood vessels and of PGCs migrating to developing gonads have been previously reported. However, the present study is the first to report on the harvest of ectopic PGCs. The defined conditions sustained continuous proliferation of tissue PGCs for at least six months and maintained PGC identity following cryopreservation. Cultured tissue PGCs of these wild species were extensively characterized for their expression of the germ cell-specific proteins, chicken vasa homolog (CVH) and deleted in azoospermia-like (DAZL), as well as the ability to colonize chicken embryonic gonads. The novel protocol is practical for generating enough PGCs for cryopreservation, transplantation, and additionally, it enables isolation of PGCs from both blood circulation and embryonic tissue simultaneously. For conservation purposes, this approach is potentially applicable more widely to other non-domestic birds than those in the family Phasianidae that were investigated in the present study.

The goat PRNT gene was initially identified as a testis-specific gene with a role in spermatogenesis. In this study, we used quantitative real-time PCR (qPCR) to first determine the mRNA expression profile of this gene in different goat tissues. Surprisingly, we found that PRNT was expressed not only in the testis but also in nine other tissues in goats. Moreover, PRNT was weakly expressed in the testis, while its expression was strongest in the ovary. These results, combined with those of other studies, led us to hypothesize that the goat PRNT gene has a role in both male and female reproduction. We further used direct DNA sequencing to detect potential SNPs within this gene in Shaanbei whit cashmere (SBWC) rams and ewes, and identified three SNPs within the PRNT gene, namely, c.-58C > T, c.71A > G (p.Alanine24Valine), and c.102C > T (synonymous). In rams, c.-58C > T and c.102C > T were strongly linked with each other (D′ = 1.000, r2 = 0.504), whereas no significant association (P > 0.05) was found between the three SNPs and semen quality, which was consistent with the low expression of the PRNT gene in the testis. Interestingly, in ewes (n = 502), c.-58C > T and c.71A > G were also strongly linked with each other (D′ = 0.973, r2 = 0.537). Additionally, the c.71A > G locus, especially the AA genotype, had a significant influence on litter size (P = 0.006), consistent with the high PRNT expression in the ovary. Combined, the results of the expression profiling and analysis of the association between the SNPs and reproductive traits showed that two strongly linked nucleotide sequence variants within PRNT were significantly associated with goat litter size. These findings provide potential DNA markers for use in the marker-assisted selection (MAS) of goats with high-fertility traits.

Copper (Cu) ions have redox activity and act as cofactors of enzymes related to respiration, radical detoxification, and iron metabolism. In this study, we aimed to examine the effects of copper (II) chloride dihydrate (CuCl2·2H2O) on porcine oocytes during in vitro maturation (IVM) and subsequent embryonic development following parthenogenetic activation (PA). Nuclear maturation, intracellular glutathione (GSH) and reactive oxygen species (ROS) levels, cumulus expansion, the mRNA expression levels of various genes, and developmental competence were analyzed. During IVM, the maturation medium was supplemented with various concentrations of Cu (0, 0.7, 1.4, and 2.8 μg/mL). After 42 h of IVM, Cu supplementation significantly increased the number of oocytes in the metaphase II stage. Further, the 1.4 μg/mL Cu group showed significantly higher intracellular GSH levels than the control group. However, Cu supplementation increased intracellular ROS levels regardless of their concentration. Additionally, the mRNA levels of Has-2, the cumulus cell expansion-related gene, were higher in all the Cu-treated groups than in the control group. The cumulus cell expansion index was higher in the 0.7 and 1.4 μg/mL Cu groups than in the other groups. In the 0.7 μg/mL Cu group, the mRNA expression levels of PCNA, Zar1, and NPM2, which are related to developmental competence, were significantly higher than those in the control group. Moreover, increased levels of Sod1 transcript, correlated with the antioxidative response, were observed in the 0.7 and 1.4 μg/mL Cu groups. The apoptosis rate in Cu-treated cumulus cells and oocytes was decreased compared to that in the corresponding control groups. Upon evaluation of subsequent embryonic development after PA, the 0.7 μg/mL Cu group showed significantly improved cleavage and blastocyst formation rate compared to the control group. In conclusion, our results suggest that Cu supplementation at appropriate concentrations in IVM medium improves porcine oocyte maturation and the subsequent embryonic potential of PA embryos by reducing oxidative stress and apoptosis.

Mycotoxins are natural contaminants of food and feed occurring worldwide. Deoxynivalenol (DON) and fumonisin B1 (FB1) are the most frequent fusariotoxins and induce immune and intestinal toxicity in humans and animals. Recently, an association between mycotoxins exposure and impaired fertility has been suggested. However, the effects of these mycotoxins on the reproductive system are not well established. This study aimed to evaluate the effects of FB1 and DON, in combination or alone, on the ovarian morphology and oxidative responses using porcine explants. Seventy-two explants were obtained from six pigs and submitted to the following treatments: control (MEM medium), DON (10 μM), FB1 (100 μM FB1), and DON + FB1 (10 μM + 100 μM). Histological and immunohistochemical assays were performed to evaluate ovarian changes, cell proliferation, and apoptosis. Oxidative stress response was evaluated through lipid peroxidation and antioxidant capacity response assays. The exposure to mycotoxins induced significant histological changes in the ovaries, which were characterized by a decrease in viable follicles and increase in degenerated follicles. A significant decrease in granulosa cell proliferation was observed in explants exposed to all mycotoxins. In addition the multi-contaminated treatment was responsible for an increase in the cell apoptosis index of growing follicles. On the other hand, the FB1 and multi-contaminated treatments induced a significant decrease in lipid peroxidation accompanied by an increase in antioxidant responses. Altogether, our results indicate a reproductive toxicity induced by fusariotoxins. Moreover, mycotoxins, alone or in combination, modulate oxidative stress response, interfering with the production of free radicals and affecting the reproductive capacity of pigs.

The objective of this study was to study the changes in salivary proteins that occur in the dog after the ejaculation process. Saliva samples from eight dogs before and after induced ejaculation were analyzed by proteomic using Tandem Mass Tag (TMT) labeling and LC-MS/MS analysis. A total of 33 salivary proteins showed significant changes after the ejaculation process. The up-regulated proteins that showed changes of higher magnitude were mucin-7 (MUC- 7), peroxiredoxin-4 (PRDX4) and galectin-3 (LEGALS3) whereas proteins such as alpha-1-acid glycoprotein (A1G1) and alpha-1B-glycoprotein (A1BG) were the most down-regulated. MUC-7 and PRDX4 expression in saliva after ejaculation could be associated with the protective “environment” created by the organism to exert pr 3o-fertility activities and antioxidants benefits in spermatozoa. Also LEGALS3 increment could be associated with an improvement of wellbeing and could contribute to a positive global effect in the body. Down-regulations of A1G1 and A1GB proteins found in saliva after ejaculation could be associated with a reduction in systemic inflammation. Overall it can be concluded that, changes in proteins in saliva that are produced after ejaculation can reflect a state of increase immune defenses, improvement of antioxidant status and low inflammation.

Fumonisin B1 (FB1), as the most toxic fumonisin, is a common Fusarium mycotoxin contaminant of feed stuff and food, posing a potential health hazard to animals and humans. FB1 has been reported to cause hepatotoxicity, neurotoxicity, nephrotoxicity, immunotoxicity and embryotoxicity; however, little information is available on whether FB1 has toxic effects on mammalian oocytes. Herein, we adopted porcine oocytes as models to explore the effects and potential mechanisms of FB1 on mammalian oocytes during in vitro maturation. Porcine cumulus oocyte complexes (COCs) were exposed to 0, 20, 30 and 40 μM FB1 for 44 h during in vitro maturation, and the results reported that first polar body (PB1) extrusion was significantly inhibited when the FB1 concentration reached 30 (P

Previous observational studies have produced strong evidence that metritis alone has an impact on reproductive performance in dairy cows; but few studies have examined the combined effect of metritis and other postpartum diseases on reproduction performance in dairy cows. Information on the combined effects of postpartum diseases on fertility in dairy cows can have practical herd-health management implications. The objective of the study reported here was to examine the observed and expected combined effects of metritis and other postpartum diseases (retained fetal membranes (RFM), clinical hypocalcemia, left displaced abomasum, clinical mastitis, lameness) on time to conception and rate of conception failure in first-lactation cows up to 150 days postpartum. The study was conducted on two commercial dairy herds in Iran. Median number of days to conception was higher in 30 cows affected with metritis and RFM (90 days), compared to 287 healthy cows (57 days), 16 cows affected with RFM alone (60 days) or 44 cows affected with metritis alone (60 days), after controlling for herd and calving month (p 0.05). In cows affected with metritis and RFM, the rate of conception failure was 3.8 times greater than that of healthy cows, after controlling for study herd and calving month (hazard ratio = 3.8; 95% CI = 2.3, 6.6; p 0.01); this observed combined effect on conception failure was two times higher than the expected combined effect based on adding or multiplying absolute independent excesses due to metritis or RFM. In addition, we observed an interaction effect between metritis and lameness on conception failure, after controlling for herd, calving month, and RFM; but study results were inconclusive. In this study, the observed combined effect of metritis and RFM on time to conception and rate of conception failure was far in excess, compared to that in cows exposed to metritis or RFM alone. Attending veterinarians or farm managers can consider this information when making breeding and culling decisions on dairy farms.

Antibiotics are added to semen extenders to control the growth of bacteria contaminating semen during collection but may contribute to the development of antibiotic resistance. An alternative would be physical separation of spermatozoa from bacteria. The objective of the present study was to evaluate two low densities of Porcicoll for removal of bacteria, and for their effect on sperm recovery and sperm quality. Semen was collected from boars at a commercial station. Aliquots of 8 extended ejaculates were subjected to colloid centrifugation through 20% Porcicoll (P20) and 30% Porcicoll (P30) in 500 mL tubes and then stored at 17 °C. Microbiological examination and sperm quality evaluation (computer assisted sperm analysis and flow cytometry) were carried out on controls and all colloid-selected samples immediately after preparation and again after storage for 3 and 7 days. The microorganisms found were mainly bacteria from the environment, gut or skin. There was a considerable reduction or complete removal of some bacteria by both colloids. Recovery rates were 86% for P20 and 81% for P30. Sperm quality was not adversely affected by colloid centrifugation on day 0, and thereafter showed a more gradual deterioration in colloid centrifuged samples than in controls, possibly due to lower bacterial contamination. There were no differences in sperm quality between the two colloid treatments. Thus, these results show that contaminating bacteria in semen can be controlled by centrifugation through low density colloids.

Oxygen concentration (02) in antral ovarian follicles is below that found in most tissues, which is important for adequate granulosa cell function. The VEGF system is linked to angiogenesis and responds to changing 02 by stimulating neovascularization when levels are low. However, in the avascular granulosa cell layer of the follicle, VEGF action is directed to stimulating cell viability and steroidogenesis. The aim of this study was to examine the effect of 02 concentration on granulosa cell expression of the VEGF-system components. Bovine granulosa cells were isolated from medium-sized follicles (4–7 mm in diameter), placed in McCoy 5a medium supplemented with 10 ng/mL of insulin, 1 ng/mL of IGF-I, and 1 ng/mL of FSH, and cultured in four well plates (500 thousand cells per well), on three separate occasions. Culture plates were placed in gas-impermeable jars with a gas mixture containing either 2%, or 5% of O2, or under atmospheric air condition inside an incubator (20% of 02). Media was replaced at 48 h of culture and cells from the plate in each oxygen concentration were pooled for RNA extraction after 96 h. The number of mRNA copies for the VEGF-system components - including ligands (VEGF120, VEGF120b, VEGF165 and VEGF165b), enzymes (cyclin-dependent like kinases-1, CLK1 and serine–arginine protein kinase 1, SRPK1), splicing factors (serine–arginine-rich splicing factors, SRSF1 and SRSF6), and the membrane-bound (VEGFR1, VEGFR2) and soluble forms of the receptors (sVEGFR1 and sVEGFR2) were quantified by qPCR. Granulosa cells cultured with low 02 (2%) had a higher expression of VEGF ligands (P 0.05). Nonetheless, mRNA copies for the soluble receptors, sVEGFR1 and sVEGFR2, linearly increased (P

The in vitro propagation and differentiation of spermatogonial stem cells (SSCs) has many potential applications within reproductive science and medicine. We established a two-dimensional (2D) cell culture system to proliferate and differentiate prepubertal mouse SSCs as a model capable of maximizing on a small number of donor SSCs. We also investigated the effects of retinol on in vitro SSC differentiation. Testis cells were cultured for 10 days in a serum-free medium. This produced SSC colonies which were then dissociated and sub-cultured for an additional 20 days in a differentiation medium. Before inducing differentiation, colonies expressed genes specific for undifferentiated spermatogonia (Ngn3, Plzf). After 10 days in the differentiation medium, Stra8 expression was upregulated. After 20 days, Acr expression was upregulated, indicating the completion of meiosis. Immunofluorescence, RT-PCR and flow cytometry confirmed the presence of haploid male germ cells (4.4% of all cells). When retinol was added to the differentiation medium the proportion of haploid germ cells increased (8.1% of cells). We concluded that, under serum-free culture conditions, prepubertal SSCs will generate colonies that can differentiate into haploid germ cells in a 2D culture system. These cells demonstrate a relatively high efficiency of haploid-cell production, which can be further improved with retinol.

Infertility is frequently associated with meiotic anomalies which can result in the production of chromosomally abnormal gametes or be concomitant with meiotic arrest. We investigated whether spermatocytes of male interspecific hybrids of the red fox (Vulpes vulpes) and the arctic fox (Alopex lagopus) presented alterations in chromosomal synapses and meiotic checkpoint signalling. Using the immunofluorescence technique with SP1 and SP3 proteins, bivalent structures and their deviations (multivalents, univalents and not fully conjugated bivalents) were analyzed on meiotic preparations. This technique allowed the localization of frequent foci of phosphorylated histones H2AHγ (Ser 139) to the meiotic block in late pachytene. These results indicate a disruption of meiotic division in male fox hybrids, which leads to a high percentage of apoptotic cells in the gonads of these animals and, consequently, sterility.

The objectives of this study were to investigate the effects of feeding 60% dried corn distillers grains plus solubles (DDGS) or the equivalent sulfur as calcium sulfate (CaSO4) on semen quality and performance characteristics in yearling bulls. Thirty-six half-sibling Angus bulls [291 ± 8.5 d; initial body weight (BW) = 320 ± 2.7 kg] were assigned to one of three diets: 1) 60% concentrate as corn (CON; S = 0.18%; n = 12); 2) 60% DDGS replacing corn (60DDGS; S = 0.55% DM; n = 12); 3) CON + equivalent sulfur of 60DDGS added as CaSO4 (SULF; S = 0.54%; n = 12). Bulls were fed for 112 d to target an average daily gain (ADG) of 1.6 kg/d. Blood samples were collected on d 0, 56, and 112, and evaluated for testosterone, thyroxine, triiodothyronine (T3) and glutathione peroxidase (GPx) activity. Ruminal H2S was measured on d 0, 14, and 42. Scrotal circumference and semen were collected on d 0, 28, 56, 84, and 112 to evaluate sperm characteristics and GPx activity in seminal plasma. A computer assisted semen analysis was used to evaluate kinematic profiles in motile and progressive sperm throughout the study. Data were analyzed as repeated measures using MIXED procedures of SAS. No differences (P ≥ 0.14) were observed for final BW, ADG, or scrotal circumference; however, SULF tended (P = 0.07) to have reduced gain:feed compared with CON, with 60DDGS being intermediate. Concentrations of ruminal H2S on d 42 were greatest (P

Flow cytometry sperm sex-sorting based on the relative DNA difference between X- and Y-chromosome bearing populations is an established method that has allowed the production of pre-sexed offspring in a multitude of species and has been a commercial success in cattle production for the past twenty years. Several improvements to the technology and the processing methods have increased the sorting efficiency of ejaculates and the post-thaw quality of sex-sorted sperm, allowing for the fertility gap between conventional (non-sorted) and SexedULTRA™ sex-sorted sperm to be bridged. Small ruminant industries are now progressively testing sex-sorted sperm for application in their specific niches and environments. A review of the key advances and the recent developments in caprine, ovine and cervine sperm sex-sorting technology are described in this publication.

The objective of this study was to evaluate the effect of two prostaglandin F2α (PGF) treatments 24 h apart (500 μg of cloprostenol) and treatment with a double PGF dose on d 7 (1000 μg of cloprostenol) during a 7-d Ovsynch protocol on progesterone (P4) concentration and pregnancy per artificial insemination (P/AI) in lactating Holstein cows. We hypothesized that treatment leads to a decreased P4 concentration at the second GnRH treatment (G2) and an increase in P/AI compared to the traditional 7-d Ovsynch protocol. A secondary hypothesis was that the treatment effect is influenced by the presence of a corpus luteum (CL) at the first GnRH treatment (G1). Two experiments were conducted on 8 commercial dairy farms in Germany. Once a week, cows from both experiments were assigned in a consecutive manner to receive: (1) Ovsynch (control: GnRH; 7 d, PGF; 9 d, GnRH), (2) Ovsynch with a double PGF dose (GDPG: GnRH; 7 d, 2xPGF; 9 d, GnRH), or (3) Ovsynch with a second PGF treatment 24 h later (GPPG: GnRH; 7 d, PGF; 8 d, PGF; 32 h, GnRH). All cows received timed AI (TAI) approximately 16 h after G2. Pregnancy diagnosis was performed by transrectal palpation (38 ± 3 d after TAI, experiment 1) or transrectal ultrasonography (35 ± 7 d after TAI, experiment 2). Whereas farms from experiment 1 used a Presynch-Ovsynch protocol (PGF, 14 d later PGF, 12 d later GnRH, 7 d later PGF, 2 d later GnRH, and 16–18 h later TAI) to facilitate first postpartum TAI, no presynchronization protocol was used on farms from experiment 2. In experiment 1, we enrolled 1581 lactating dairy cows (60 experimental units) from 2 dairy farms. At G2, blood samples were collected from a subsample of cows (n = 491; 16 experimental units) to determine P4 concentration at G2. In experiment 2, we enrolled 1979 lactating dairy cows (252 experimental units) from 6 dairy farms. Transrectal ultrasonography was performed to determine the presence or absence of a CL at G1. In experiment 1, treatment affected P/AI (P = 0.01) and P/AI was greater for GDPG (38.2%) and GPPG (38.9%) than for control cows (29.8%). Both, GDPG and GPPG cows had decreased P4 concentration at G2 compared with control cows (P

Introduction A decline in semen quality caused by global warming and torrid working conditions is a major cause of human male infertility, and heat stress-induced decreases in male reproductive ability results in economic losses in livestock husbandry. Increasing evidence suggests that melatonin exerts protective effects on stress-induced DNA damage and apoptosis in germ cells. However, few studies have assessed the effects of melatonin on testicular recovery during post-heat stress and the underlying mechanisms. Methods and results In vivo studies using 8-week-old male CD-1 mice revealed that melatonin pretreatment (50 mg/kg for 5 days) did not alleviate heat stress-induced germ cell loss and disrupted testicular histomorphology, however, long-term melatonin administration after heat stress accelerated germ cell apoptosis, spermatogenic cell regeneration, and testicular weight recovery. In vitro studies demonstrated that melatonin enhanced RAC1 activity, resulting in increased phagocytosis of apoptotic germ cells by Sertoli cells. In addition, melatonin restored gap junctions and tight junctions after heat stress, thereby promoting hollow seminiferous tubule filling. Discussion Long-term melatonin administration accelerated testicular recovery after heat stress by enhancing the phagocytotic activity of Sertoli cells and the regeneration of spermatogenic cells. This finding suggests that melatonin is a potential therapeutic for heat stress-induced male infertility.

Heat stress has been widely studied in relation to its effects on the production and reproduction of dairy cattle. However, the long-term effects of heat stress during intrauterine development on adult cows have been scarcely considered. Thus, the aim of this study was to evaluate possible changes in the reproductive performance of Holstein cows gestated under different values of the Temperature-Humidity Index (THI) during their intrauterine development. Data collected from a database of reproductive and productive records of 10,790 Holstein cows from the central region of Argentina and the THI data from the agrometeorological station of the Instituto Nacional de Tecnología Agropecuaria (INTA) EEA Rafaela, Santa Fe, Argentina, were used. The gestation of the cows evaluated was divided into trimesters, in which the highest THI cycles (sum of three or more consecutive days of exposure to a THI ≥72 during each trimester) and number of days with a THI ≥ 72 were calculated. The calving-to-conception and calving-to-first-service intervals of the cows evaluated were considered as reproductive variables associated with their first lactation. Generalized Linear Mixed Models were used, considering the cow's dairy farm as a random factor within the model. The exposure of the animals to environments with a THI ≥72 during the first trimester of gestation had a negative impact on the reproductive efficiency parameters analyzed. The results obtained indicate that the exposure of pregnant females to high THI values has a long-term impact on their daughters, which may contribute to a decrease in their reproductive performance, possibly through inherited epigenetic characteristics that remain in later generations through fetal programming.

The objective of this study was to evaluate the effect of the administration of estradiol cypionate (ECP) at the end of an estradiol and progesterone-based protocol for fixed time artificial insemination (FTAI) on ovarian response and uterine function in postpartum anestrous beef cows. Multiparous suckled cows were randomly assigned to receive ECP at doses of 0 (control, n = 15), 0.5 (n = 15) or 1.0 mg (n = 15) im at the time of progesterone intravaginal insert removal. Serum 17β-estradiol concentrations at 24 h after insert removal were greater (P 0.05) in both ECP treatments than in controls. No differences in estradiol were found between 0.5 mg and control cows (P 0.1) from 48 h after insert removal until ovulation, although greater (P 0.05) concentrations were maintained until ovulation in 1.0 mg ECP treated cows. Maximum 17β-estradiol concentration attained in each female was greater as ECP dose was greater (10.4 ± 0.4, 11.8 ± 0.5 and 13.5 ± 0.7, for control, 0.5 and 1.0 mg ECP treated cows, respectively; P 005). Proportion of cows that ovulated tended to be greater (P = 0.06) in ECP treated than in control cows. Ovulation occurred earlier and the size of the ovulatory follicle was smaller (P 0.05) for 1.0 mg but not for 0.5 mg (P 0.1) when compared with control cows. After ovulation (Day 13 and 14), serum progesterone concentrations were greater (P 0.05) in 0.5 and 1.0 mg ECP than control cows. Uterine environment on Day 6 after ovulation was affected by treatment; transcript expression of three of nine evaluated genes (i.e., estrogen, IGF-1 and insulin receptors genes) were upregulated (P 0.05) after ECP treatment. In conclusion, ECP administration at progesterone insert removal in anestrous cows i) induces greater serum estradiol concentrations and tended to induce greater ovulation rate, ii) acts in a dose-dependent manner, as ECP dose increases ovulation occurs earlier and the size of the ovulatory follicle is smaller, iii) improves postovulatory luteal function and affects uterine gene expression. Altogether, this information contributes with the understanding of the effect of preovulatory estradiol exposure on ovulation and postovulatory ovarian and uterine function in anestrous beef cows.

The spermicidal effects of silver nanoparticles (AgNPs) hinder its application in the field of artificial insemination. In this study, silver-carbon NPs (Ag@C NPs) was synthesized and applied as an alternative antibiotic agent for bull semen extender. Ag@C NPs were characterized using X-ray diffraction (XRD), X-ray photoelectron spectroscopy (XPS), atomic absorption flame spectroscopy, transmission electron microscope (TEM), and high-resolution TEM (HR-TEM). Data analysis revealed the successful synthesis of Ag@C NPs with a particle size of 1–5 nm (average particle size of 2.5 nm) embedded into carbon. The antimicrobial activity of Ag@C NPs was tested against bacteriospermia of fresh semen collected from five fertile bulls (three ejaculates/bull). Escherichia coli (E. Coli), Staphylococcus aureus (S. aureus), and Pseudomonas aeruginosa (P. aeruginosa) were isolated from fresh semen samples and identified by culture, staining, and conventional biochemical tests. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of Ag@C NPs against bacteriospermia was determined at 5 and 37 °C. Ag@C NPs showed efficient antimicrobial activity (MIC: 3.125–12.5 μg/mL) against the tested strains and strong bactericidal effect on S. aureus, and P. aeruginosa (MBC: 3.125 μg/mL), with no detrimental effect (P ˃ 0.05) on the percentage of sperm motility (70.71 ± 4.82; 74.65 ± 4.46), plasma membrane integrity (68.39 ± 4.31; 72.38 ± 4.91), acrosome integrity (88.40 ± 13.21; 86.77 ± 14.23), and normal sperm morphology (86.85 ± 7.43; 87.82 ± 8.15) at concentrations of 15 and 30 μg/mL, respectively, after a cold storage of 48 h. However, Ag@C NPs showed a detrimental effect on sperm parameters in a dose dependent manner at concentrations ≥60 μg/mL. Ag@C NPs showed no adverse effect on the sperm’s ultrastructure with limited sperm internalization at MIC. In conclusion, Ag@C NPs could be used as an alternative antibiotic agent for bull semen extender without a significant cytotoxic effect on the sperm during cold storage. However, further investigations for their effects on embryo production and female genitalia are still required.

Zonula occludens (ZO)-1 and ZO-2 are involved in epithelial polarity maintenance, gene transcription, cell proliferation and tumor cell metastasis. Regulating ZO-1/2 expression influences the early embryonic development of mice, but whether they are involved in oocyte maturation is still poorly understood. In the present study, the expression patterns of ZO-1 and ZO-2 in porcine cumulus cells and oocytes matured in vitro and early embryos from parthenogenetic activation were detected by qRT-PCR or Western blot, and then their roles in porcine oocyte maturation and early embryo development were investigated by shRNA technology. ZO-1 and ZO-2 were found to be expressed in cumulus cells, oocytes and early embryos, while ZO-1α+ was expressed only in cumulus cells, morula and blastocysts. During in vitro maturation (IVM), the abundance of ZO-1 and ZO-2 in oocytes was significantly higher than that in cumulus cells at 0 h (P 0.05). However, the blastocyst rate was reduced by treatment of COCs with ZO-1-shRNA1 but not ZO-2-shRNA2. The total cell number of blastocysts was decreased by downregulation of ZO-1 and ZO-2 (P 0.05). Altogether, the above results indicate that ZO-1 and ZO-2 display similar expression patterns during porcine oocyte IVM and are critical to porcine oocyte maturation and early embryonic development.