Maternal nutrition and fetal imprinting of the male progeny.

The global population as well as the demand for human food is rapidly growing worldwide, which necessitates improvement of efficiency in livestock operations. In this context, environmental factors during fetal and/or neonatal life have been observed to influence normal physical and physiological function of an individual during adulthood, and this phenomenon is called fetal or developmental programming. While numerous studies have reported the impact of maternal factors on development of the female progeny, limited information is available on the potential effects of fetal programming on reproductive function of the male offspring. Therefore, the objective for this review article was to focus on available literature regarding the impact of maternal factors, particularly maternal nutrition, on reproductive system of the male offspring. To this end, we highlighted developmental programming of the male offspring in domestic species (i.e., pig, cow and sheep) as well as laboratory species (i.e., mice and rat) during pregnancy and lactation. In this sense, we pointed out the effects of maternal nutrition on various functions of the male offspring including hypothalamic-pituitary axis, hormonal levels, testicular tissue and semen parameters.

Developmental programming of production and reproduction in dairy cows: IV. Association of maternal milk fat and protein percentage and milk fat to protein ratio with offspring's birth weight, survival, productive and reproductive performance and AMH concentration from birth to the first lactation period.

Although the association of maternal milk production with developmental programming of offspring has been investigated, there is limited information available on the relationship of maternal milk components with productive and reproductive performance of the offspring. Therefore, the present study was conducted to analyze the association of maternal milk fat and protein percentage and milk fat to protein ratio with birth weight, survival, productive and reproductive performance and AMH concentration in the offspring. In study I, data of birth weight, milk yield and reproductive variables of offspring born to lactating dams (n = 14,582) and data associated with average maternal milk fat percentage (MFP), protein percentage (MPP) and fat to protein ratio (MFPR) during 305-day lactation were retrieved. Afterwards, offspring were classified in various categories of MFP, MPP and MFPR. In study II, blood samples (n = 339) were collected from offspring in various categories of MFP, MPP and MFPR for measurement of serum AMH. Maternal milk fat percentage was positively associated with birth weight and average percentage of milk fat (APMF) and protein (APMP) and milk fat to protein ratio (FPR) during the first lactation, but negatively associated with culling rate during nulliparity in the offspring (P < 0.05). Maternal milk protein percentage was positively associated with birth weight, APMF, APMP, FPR and culling rate, but negatively associated with milk yield and fertility in the offspring (P < 0.05). Maternal FPR was positively associated with APMF and FPR, but negatively associated with culling rate, APMP and fertility in the offspring (P < 0.05). However, concentration of AMH in the offspring was not associated with MFP, MPP and MFPR (P > 0.05). In conclusion, the present study revealed that maternal milk fat and protein percentage and their ratio were associated with birth weight, survival, production and reproduction of the offspring. Yet it was a preliminary research and further studies are required to elucidate the mechanisms underlying these associations.

Developmental programming of reproduction in the female animal.

Successful reproduction is a cornerstone in food animal industry in order to sustain food production for human. Therefore, various methods focusing on genetics and postnatal environment have been identified and applied to improve fertility in livestock. Yet there is evidence indicating that environmental factors during prenatal and/or neonatal life can also impact the function of reproductive system and fertility in the animals during adulthood, which is called the developmental programming of reproduction. The current review summarizes data associated with the developmental origins of reproduction in the female animals. In this regard, this review focuses on the effect of plane of nutrition, maternal body condition, hypoxia, litter size, maternal age, parity, level of milk production and milk components, lactocrine signaling, stress, thermal stress, exposure to androgens, endocrine disrupting chemicals, mycotoxins and pollutants, affliction with infection and inflammation, and maternal gut microbiota during prenatal and neonatal periods on the neuroendocrine system, puberty, health of reproductive organs and fertility in the female offspring. It is noteworthy that these prenatal and neonatal factors do not always exert their effects on the reproductive performance of the female by compromising the development of organs directly related to reproductive function such as hypothalamus, pituitary, ovary, oviduct and uterus. Since they can impair the development of non-reproductive organs and systems modulating reproductive function as well (e.g., metabolic system and level of milk yield in dairy animals). Furthermore, when these factors affect the epigenetics of the offspring, their adverse effects will not be limited to one generation and can transfer transgenerationally. Hence, pinpointing the factors influencing developmental programming of reproduction and considering them in management of livestock operations could be a potential strategy to help improve fertility in food animals.

Developmental programming of the ovarian reserve in livestock.

Mammalian females are born with a finite number of follicles in their ovaries that is referred to as the ovarian reserve. There is a large amount of variation between females in the number of antral follicles that they are born with, but this number is positively correlated to size of the ovarian reserve, has a strong repeatability within a female, and a moderate heritability. Although the heritability is moderate, numerous external factors including health, nutrition, ambient temperature, and litter size influence the size and function of the ovarian reserve throughout life. Depletion of the ovarian reserve contributes to reproductive senescence, and genetic and epigenetic factors can lead to a more rapid decline in follicle numbers in some females than others. The relationship of the size of the ovarian reserve to development of the reproductive tract and fertility is generally positive, although some studies report antagonistic associations of these traits. It seems likely that management decisions and environmental factors that result in epigenetic modifications to the genome throughout life may cause variability in the function of ovarian genes that influence fecundity and fertility, leading to differences in reproductive longevity among females born with ovarian reserves of similar size. This review summarizes our current understanding of factors influencing size of the ovarian reserve in cattle, sheep, and pigs and the relationship of the ovarian reserve to reproductive tract development and fertility. It provides strategies to apply this knowledge to improve diagnostics for better assessment of fertility and reproductive longevity in female livestock.

Developmental programming of production and reproduction in dairy cows: III. Association of level of maternal milk production with offspring's birth weight, survival, productive and reproductive performance and AMH concentration from birth to the first lactation period.

Although some studies investigated the relationship of dam milk production (DMP) with offspring birth weight and productive performance, limited information is available on the association of level of DMP with reproductive performance in dairy cows. Therefore, the present study was conducted to understand whether dams with various levels of milk production produce offspring with different fertility. In study I, offspring were classified based on the level of DMP into five categories, including DMP1 (dams with <10.00 × 103 kg of 305-day milk production), DMP2 (dams with ≥10.00 × 103 kg and <12.00 × 103 kg of 305-day milk production), DMP3 (dams with ≥12.00 × 103 kg and <14.00 × 103 kg of 305-day milk production), DMP4 (dams with ≥14.00 × 103 kg and <16.00 × 103 kg of 305-day milk production) and DMP5 (dams with ≥16.00 × 103 kg of 305-day milk production). In study I, data of birth weight, milk yield and reproductive variables of 14,536 offspring born to lactating dams and corresponding data of DMP were retrieved. In study II, blood samples (n = 339) were collected from offspring in various categories of DMP for measurement of serum AMH. Offspring were heavier at birth in DMP4 and DMP5 categories than DMP1 and DMP2 categories (P < 0.05). Milk yield of offspring increased as DMP elevated (P < 0.05); however, offspring in DMP1 and DMP2 categories produced higher milk as compared with their dams during primiparity (P < 0.05) whereas offspring in DMP3, DMP4 and DMP5 categories produced less milk as compared with their dams during primiparity (P < 0.05). Milk fat to protein ratio during the first month of lactation was greater in DMP4 and DMP5 categories than DMP1 category (P < 0.05). Offspring of DMP4 and DMP5 categories were inseminated and conceived at younger ages than offspring of DMP1 category during nulliparity (P < 0.05). Calving to conception interval was longer in DMP5 than DMP1 category in primiparous offspring (P < 0.05), but concentration of AMH did not differ among various categories of DMP (P > 0.05). In conclusion, dams with greater level of milk production produced heavier offspring with higher milk yield but worse transgenerational improvement in milk production and diminished reproductive performance, which were seemingly under higher pressure of negative energy balance during the first month of lactation.

Oocyte Vitrification Reduces its Capability to Repair Sperm DNA Fragmentation and Impairs Embryonic Development.

Oocytes play a crucial role in repairing sperm DNA damage, which can affect the next generation; however, certain factors can impair this ability. This study examined whether oocyte vitrification, a widely used method for fertility preservation, negatively affects repair ability. Male DBA/2 mice (n = 28) were injected with 101.60 µmol/100 g body weight of tert-Butyl hydroperoxide (tBHP) for 14 days to induce sperm DNA damage. Histological changes, sperm functions, and DNA fragmentation were assessed using the TUNEL assay. Cumulus-oocyte-complexes (COCs) of superovulated female DBA/2 mice (n = 28) were vitrified using the Cryotop method. Fresh and vitrified oocytes were then fertilized by tBHP-treated and untreated sperms, and subsequent embryonic development was monitored. Additionally, the expression of Mre11a, Rad51, Brca1, and Xrcc4 was assessed in resulting zygotes and blastocysts using real-time PCR. The sperm tBHP treatment reduced differentiated spermatogenic cells in the testicular tissue, sperm concentration, and motility, while increasing DNA fragmentation (P < 0.05). The fertilization rate was decreased in the tBHP-treated sperm-vitrified oocyte group (P < 0.05), and the two-cell rate diminished in tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05). The four-cell to blastocyst rate decreased in the untreated sperm-vitrified oocyte and the tBHP-treated sperm-fresh and vitrified oocyte groups (P < 0.05), and the tBHP-treated sperm-vitrified oocyte groups had the lowest blastocyst rate. In zygotes, Brca1 was upregulated in the tBHP-treated sperm-vitrified oocyte group (P < 0.05). Also, in blastocysts, Rad51, Brca1, and Xrcc4 were significantly upregulated in the untreated sperm-vitrified oocytes group (P < 0.05). Damages to the oocyte due to vitrification can disrupt the repair of sperm DNA fragmentation and consequently impair the embryo development.

Evaluation of the effect of melatonin implantation in rams and eCG dose in ewes synchronized by a CIDR-eCG protocol on reproductive performance of Lacaune sheep breed during non-breeding season.

Sustainable production of milk is favorable in dairy sheep industry, which necessitates year-round reproduction of rams and ewes even during non-breeding season. Hence, protocols facilitating reproduction of rams and ewes during non-breeding season are of importance for this purpose. Therefore, the present study was conducted to investigate the effect of melatonin implantation in rams and administration of 400 IU eCG (E400) versus 300 IU eCG (E300) in ewes on reproductive performance of Lacaune sheep breed during non-breeding season. Rams were allocated to two groups including untreated (control, CON; n = 36) and melatonin-treated (MEL; n = 37). A subset of rams from CON (n = 7) and MEL (n = 7) groups were used for assessment of scrotal circumference, kinematic and functional characteristics of sperm, total antioxidant capacity (TAC) of semen and circulating testosterone on Days 0 (the beginning of study) and 60 (60 days after melatonin implantation). Further, the study had a 2 × 2 factorial design with four experimental groups including 1) ewes treated with E300 and introduced to CON rams (E300CON; n = 17 rams and 172 ewes), 2) ewes treated with E300 and introduced to MEL rams (E300MEL; n = 18 rams and 177 ewes), 3) ewes treated with E400 and introduced to CON rams (E400CON; n = 19 rams and 192 ewes), and ewes treated with E400 and introduced to MEL rams (E400MEL; n = 19 rams and 190 ewes). Melatonin implantation improved scrotal circumference, concentration, progressive motility, velocity, mitochondrial membrane potential and viability of sperm, TAC of semen, concentration of testosterone and fertility of rams (P < 0.05). Besides, E400 compared with E300 enhanced synchronization of estrus and fertility in ewes (P < 0.05). In conclusion, melatonin implantation promoted reproductive performance in Lacaune rams, and increase in dose of eCG improved reproductive performance in Lacaune ewes.

Optimisation of hormonal treatment to improve follicular development in one-day-old mice ovaries cultured under in vitro condition.

CONTEXT: Base medium containing knock-out serum replacement (KSR) has been found to support formation and maintenance of follicles in one-day-old mice ovaries, but has not been shown to properly support activation and growth of primordial follicles. AIMS: The present study was conducted to tailor the hormonal content of base medium containing KSR to enhance development of primordial follicles in neonatal ovaries. METHODS: One-day-old mice ovaries were initially cultured with base medium for four days, and then, different hormonal treatments were added to the culture media and the culture was proceeded for four additional days until day eight. Ovaries were collected for histological and molecular assessments on days four and eight. KEY RESULTS: In experiment I, the main and interactive effects of FSH and testosterone were investigated and FSH promoted activation of primordial follicles and development of primary and preantral follicles, and upregulated genes of phosphoinositide 3-kinase (Pi3k ), KIT ligand (Kitl ), growth differentiation factor 9 (Gdf9 ) and follicle stimulating hormone receptor (Fshr ) (P Bmp15 ), Connexin-43 (Cx43 ) and luteinising hormone and choriogonadotropin receptor (Lhcgr ) (P P Lhcgr (P P >0.05). CONCLUSIONS: Supplementation of culture medium containing KSR with gonadotropins, particularly hMG, could improve follicular growth and expression of factors regulating follicular development. IMPLICATIONS: This study was a step forward in formulating an optimal medium for development of follicles in cultured one-day-old mice ovaries.

Developmental programming of production and reproduction in dairy cows: II. Association of gestational stage of maternal exposure to heat stress with offspring's birth weight, milk yield, reproductive performance and AMH concentration during the first lactation period.

Although the negative effect of maternal exposure to heat stress on production and reproduction of offspring has been reported, there are some discrepancies among various studies about which gestational stage is more critical in this regard. Therefore, the present research was conducted to identify during which stage(s) of pregnancy maternal exposure to heat stress would lead to more dramatic decrease in productive and reproductive performance of offspring. To this end, offspring were classified based on the gestational stage they were in utero exposed to heat stress into four categories, including heat stress exposure (HSE) during only the first trimester of gestation (HSE1), HSE during the first and second trimester of gestation (HSE2), HSE during the second and third trimester of gestation (HSE3) and HSE during only the third trimester of gestation (HSE4). In study I, data of birth weight, milk yield and reproductive variables of 11,788 offspring and data of the month they were conceived were retrieved. In study II, blood samples (n = 521) were collected from offspring in various categories of HSE for measurement of serum AMH. Offspring in HSE1 and HSE2 categories were heavier than offspring in HSE3 and HSE4 categories (P < 0.0001). Offspring in HSE1 and HSE3 categories had the lowest and highest milk production, respectively (P < 0.05). First service conception rate was the greatest and worst in HSE1 and HSE4 categories, respectively (P < 0.05). Service per conception and calving to conception interval were greater in HSE2 than HSE4 category (P < 0.05). Concentration of AMH was lower in HSE1 than HSE4 category (P < 0.05). In conclusion, the present study indicated that the early stage of gestation could be a more critical period for the negative impact of in utero heat stress on developmental programming of milk production and ovarian reserves. Yet an evident temporal pattern for the adverse effect of maternal heat stress on developmental programming of reproductive performance in offspring was not found.

Successful 3D culture and transplantation of mouse isolated preantral follicles in hydrogel of bioengineered Wharton's jelly.

MAIN OBJECTIVE: Due to Human Wharton's Jelly (HWJ) could be applied in tissue engineering as a bio scaffold, the present study was conducted to investigate the effects of HWJ hydrogel on in vitro culture and auto-transplantation of mouse ovarian follicles. MATERIALS AND METHODS: HWJ was isolated from umbilical cord and decellularized with SDS/Tris/EDTA. DNA, Collagen and Glycosaminoglycans (GAGs) were measured. Decellularized Wharton's Jelly (DWJ) was dissolved to make Wharton's Jelly Hydrogel (WJH), and composited with Alginate (ALG) (1.5%) in equal ratio (WJH+ALG). Then, mouse preantral follicles were isolated and encapsulated in 10µL droplets of WJH and randomly considered for both 14 days culture and auto-transplantation. RESULTS: Collagen, GAGs and DNA evaluations showed majority of WJ cells have been removed and MTT approved no toxicity. Degradation rate and rheological analysis represented optimal hydrogel compatibility. The data from in vitro culture revealed significant antral formation in WJH+ALG (P≤0.05). In transplantation, follicles failed to survive in ALG; however, survived in WJH+ALG to antral stage (P<0.05). VEGF and CD34 had greater expression in WJH+ALG than ALG (P< 0.05). CONCLUSION: Wharton's jelly hydrogel and Alginate compound is interesting composite for successful development of mouse preantral follicles in both 3D in vitro culture and transplantation.

The study of a hermaphroditic sheep caused by a mutation in the promoter of SRY gene.

In mammals, sex-determining region Y (SRY) gene plays vital role as a transcription factor to regulate the expression of the genes contributing to development of male genitals. Any mutation disrupting expression of SRY gene can cause disorders of sex development (DSDs). In this study, the examination of a hermaphroditic (female-like) Shal sheep which was referred for infertility is described. Initially, the reproductive system of the sheep was histologically and anatomically assessed. Karyotyping was used to determine the real gender of the animal. Sex hormones including progesterone, estradiol, and testosterone were measured by enzyme-linked immunosorbent assay (ELISA). Eventually, promoter part and SRY gene were sequenced and aligned to detect any potential mutation using NCBI data base. Although anatomical inspection led to identification of uterus, ovary, and enlarged clitoris as well as testes in the sheep, the karyotyping results interestingly revealed that the animal was genetically a male. Although the sheep had both male and female gonads, there were no overt signs of reproductive behavior and gamete production was not observed. Plasma steroid hormone levels were reported to be at basal levels. Additionally, a mutation was detected on the promoter of the SRY gene. In conclusion, the case implies that mutation on the promoter part of SRY gene could disrupt sexual development of the fetus culminating in DSDs in the sheep.

Developmental programming of production and reproduction in dairy cows: I. Association of maternal parity with offspring's birth weight, milk yield, reproductive performance and AMH concentration during the first lactation period.

Multiparous dams have been reported to produce offspring with greater fertility and higher AMH concentration, as a marker of ovarian reserves, as compared with nulliparous and primiparous dams. Yet it has remained to be addressed whether this phenomenon can still be true for old multiparous cows which might experience some geriatric changes in their reproductive system. Therefore, the present study was conducted to evaluate the productive and reproductive performance of offspring with different maternal parity. To this end, offspring were classified based on their maternal parities into four categories, including offspring of nulliparous (no previous parity), primiparous (one previous parity), young multiparous (two to six previous parities) and old multiparous (seven or more previous parities) dams. In study I, data of birth weight, milk yield and reproductive variables of 11,788 offspring and data of their maternal parity were retrieved. In study II, blood samples (n = 521) were collected from offspring with various maternal parity for measurement of serum AMH. Birth weight was the lowest in the offspring of nulliparous dams (P < 0.0001) and it was lower in offspring of primiparous and old multiparous dams than offspring of young multiparous dams (P < 0.05). Milk production was the lowest in offspring of old multiparous dams (P < 0.01), and it was lower in offspring of young multiparous dams than offspring of nulliparous and primiparous dams (P < 0.0001). Offspring of old multiparous dams had greater first service conception rate, less services per conception and shorter calving to conception interval than offspring of nulliparous, primiparous and young multiparous dams (P < 0.05). Furthermore, AMH concentration was higher in offspring of old multiparous dams than offspring of nulliparous and primiparous dams (P < 0.05). In conclusion, the present study revealed greater milk production in offspring resulting from dams with lower parity, probably due to the genetic selection for improvement of milk production in dairy cows which imparts the younger generations greater genetic merits for milk production. Reproductive performance, however, was greater in offspring born to dams with higher parity, particularly those born to old multiparous dams, and this phenomenon might be related to their lower milk production and higher AMH concentration.

Replacement of the first GnRH by estradiol in the breeding Ovsynch of Double Ovsynch protocol could improve fertility in Holstein dairy cows.

Unlike GnRH, estradiol could induce emergence of a new follicular wave regardless of the size of follicle. Therefore, the present study was conducted to understand whether replacement of the first GnRH by estradiol in the breeding protocol of Double Ovsynch program could enhance fertility. Cows were randomly assigned to two groups, including Double Ovsynch protocol (Control; n = 120) and Ovsynch-estradiol-PGF2α-GnRH (EPG) protocol (Treatment; n = 120). Cows in both groups were subjected to presynchronization Ovsynch. Seven days later, cows in the control group received GnRH, which was followed by PGF2α and GnRH 7 days and 9 days plus 8 h later, respectively. Cows in treatment group received estradiol 7 days after the second GnRH of presynchronization Ovsynch, which was followed by PGF2α and GnRH 7 days and 10 days plus 8 h later, respectively. Cows were subjected to timed AI (TAI) 16 h after final GnRH in both groups. Pregnancy per AI (P/AI) was greater in cows in treatment than control group (64.17 % vs. 44.17 %, respectively; P = 0.02). Cows with a follicle with diameter ≥ 10 mm (F10) at the beginning of EPG in treatment group had greater P/AI than cows without a F10 at the beginning of breeding Ovsynch in control group (P ≤ 0.05). Pregnancy per AI was greater in cows with a CL at the beginning of EPG in treatment group than cows without a CL at the same timepoint in treatment group, and cows with or without a CL at the beginning of breeding Ovsynch in control group (P ≤ 0.05). In conclusion, inclusion of estradiol in Double Ovsynch protocol as a replacement for the first GnRH of breeding Ovsynch could improve fertility, particularly in cows with a CL at the initiation of EPG.

Evidence that elevation of maternal somatic cell count could lead to production of offspring with inferior reproductive and productive performance in dairy cows during the first lactation period.

Although the effect of mastitis on reproduction and production of lactating dairy cows has been vastly studied, little information is available about the association of maternal udder health status with offspring reproduction and production. Therefore, the present research was conducted to study the association between maternal average monthly somatic cell count (SCC) with reproduction, anti-Müllerian hormone (AMH) concentration, udder health status and milk production in the offspring. Based on maternal average monthly SCC (MSCC), offspring were classified into five categories including MSCC1 (SCC <200,000; n = 3005), MSCC2 (200,000 ≤ SCC <400,000; n = 252), MSCC3 (400,000 ≤ SCC <600,000; n = 103), MSCC4 (600,000 ≤ SCC <800,000; n = 40) and MSCC5 (SCC ≥800,000; n = 61). Data associated with reproduction, production and udder health status of offspring were retrieved from the herd database. In addition, blood samples were collected from a subset of offspring (n = 136) for measurement of serum AMH, as a reliable marker of ovarian reserves. Offspring in MSCC5 category had more services per conception and longer calving to conception interval than offspring in MSCC1 and MSCC2 categories (P < 0.05). The average number of SCC and risk of clinical mastitis in the offspring were not associated with MSCC (P > 0.05). But offspring in MSCC5 category produced less milk, fat and protein than offspring in MSCC1 category (P < 0.05). In addition, AMH concentration was lower in MSCC5 than MSCC1 category (P < 0.05). In conclusion, the present study showed that elevated maternal average monthly SCC could culminate in birth of offspring with inferior reproductive performance, smaller size of ovarian reserves and lower level of milk production during the first lactation period.

Effect of Mechanical Micro-Vibrations on The Efficiency of Leopard Inter-Species Somatic Cell Nuclear Transfer.

OBJECTIVE: Scarcity of oocytes for assisted reproduction in endangered species can be bypassed by interspecies somatic cell nuclear transfer (iSCNT). In Felids, domestic cat (Felis catus) oocytes can serve as recipients for the nucleus of the endangered Persian leopard (Panthera pardus saxicolor). However, in vitro oocyte maturation is still suboptimal in cats, whereas it has been reported to benefit from micro-vibration in non-felid species. Therefore, the present study is aimed to determine whether micro-vibration, applied during in vitro maturation (IVM), improves the embryogenic potential of cat oocytes transplanted with fibroblast nuclei of the Persian leopard. MATERIALS AND METHODS: In the experimental study, cat cumulus-oocyte complexes (COCs) were randomly assigned to the treatment group (micro-vibration) or control group (static culture). Resultant metaphase II (MII) oocytes were enucleated and reconstructed with nucleus transplants from leopard fibroblasts, followed by artificial oocyte activation and embryo culture under the same condition (static) for 7 days. RESULTS: While cumulus cell expansion and oocyte maturation profited from micro-vibration (P<0.05), the quantity and quality of blastocysts were significantly lower in micro-vibration than in the control group (P<0.05). The total number of blastocyst cells tended to be lower in the micro-vibration than in the control group (P=0.075). Nevertheless, the proportion of ICM and TE cells did not differ between the micro-vibration and control groups (P>0.05). CONCLUSION: The present study indicated that micro-vibration at a frequency of 44 Hz for 5 secs per hour enhanced nuclear maturation and cumulus cell expansion of cat oocytes. However, exposure to micro-vibration during IVM impaired the survival rate of reconstructed oocytes during the iSCNT process and their developmental competence toward the blastocyst stage.

Evidence that Murciano-Granadina does with longer anogenital distance are more fertile and prolific and produce heavier and male-biased litters.

Anogenital distance (AGD) is related to fertility in cows, but there is little information on the association of AGD and reproduction in does. Therefore, the present study was conducted to assess the relationship between AGD and reproductive variables in Murciano-Granadina does. AGD was measured as the distance between anus and clitoris and does (n = 578) were divided into two categories of AGD, including short AGD (AGD ≤ median of AGD in studied does; n = 313) and long AGD (AGD > median of AGD in studied does; n = 265). Data of reproductive variables were retrieved from the herd database and were analyzed using multivariable statistical models, in which the potential confounding factors were included. AGD data were not normally distributed (3.78 ± 0.02 cm) and ranged from 2.70 to 5.10 cm. AGD was longer in multiparous and primiparous does than nulliparous does (P < 0.0001), and was positively associated with age and body weight (P < 0.0001). The risk of pregnancy and kidding, litter size, fecundity, proportion of male offspring and birth weight of kids were higher in long AGD does than short AGD does (P < 0.05). But abortion risk did not differ between does with long and short AGD (P > 0.05). In conclusion, the present study revealed that AGD had individual variation among Murciano-Granadina does, and does with long AGD were more fertile and more likely to produce larger, heavier and male-biased litters as compared with does with short AGD.

Effects of N-Acetyl-L-Cystein Antioxidant on Ex Vivo Culture of Vitrified Premature Mouse Ovarian Tissue.

Optimization of practical ways to obtain mature follicles from cryopreserved ovarian tissues, especially in patients suffering from ovarian dysfunction, is very important. In vitro ovarian tissue culture allows faster screening of follicle development and reduces follicle isolation damage. During ovarian tissue culture, controlling oxidative stress is critical to support better follicular development and less damage. Immature Naval Medical Research Institute (NMRI) mouse ovaries (8-days-old) were randomly distributed into four cultured groups; non-vitrified, vitrified, non-vitrified N-acetyl-L-cysteine (NAC)+, and vitrified NAC+. Ovaries of vitrified groups along with non-vitrified ovaries were cultured on agar gel in the presence or absence of NAC for 5 days. Afterward, morphological evaluations, mRNA expressions of Gdf9, Bmp6, Lif, Amh, Bax, and Bcl2 genes, malondialdehyde, and total antioxidant capacities were compared between four groups at the first and last day of culture. Good preservation of tissue integrity and an increase of follicular development were observed in all groups. In addition, the expression of Gdf9, Lif, Bax, and Bcl2 genes were increased and Amh was decreased in groups cultured in the presence of NAC compared to groups cultured without NAC. Although total antioxidant capacity was not significantly different between the experimental groups, the lipid peroxidation and apoptotic index were significantly reduced in the presence of NAC. Thus, it appears that NAC antioxidant acts as a contributory factor for the ex vivo culture of ovarian tissue and reduces oxidative stress, apoptotic index, and improves follicular development, especially in non-vitrified groups.

High steroid content in conditioned medium of granulosa cells may disrupt primordial follicles formation in in vitro cultured one-day-old murine ovaries.

This study was conducted to investigate the main and interactive effects of two methods of culture medium preparation [base medium vs granulosa cells conditioned medium (GCCM)] and two nutrient supplements [fetal bovine serum (FBS) vs knock-out serum replacement (KSR)] on formation and activation of primordial follicles and gene expression of corresponding factors during a seven-day culture period. One-day-old mouse ovaries were cultured with four different culture media including base medium containing FBS (BMF), base medium containing KSR (BMK), GCCM prepared with FBS (CMF) and GCCM prepared with KSR (CMK), and samples for histological and molecular assessments were collected on days 3 and 7 of culture. Further, steroid content of media was measured. Histological examination showed that KSR enhanced follicular formation and the number of follicular count was greater in BMK than CMF group (P < 0.05). Moreover, follicular activation was higher in CMK group than BMK and CMF groups (P < 0.05). Additionally, RT-PCR revealed that KSR upregulated Gdf9 gene expression (P < 0.05), while GCCM diminished expression of Gdf9, Bmp15, Notch2, Figla and Foxl2 (P < 0.05). GCCM decreased expression of Pten and increased expression of Pi3k (P < 0.05). Besides, hormonal assays indicated higher concentrations of estradiol and progesterone in GCCM compared with base media (P < 0.0001). In conclusion, the present study showed base medium containing KSR could serve as a proper medium for in vitro culture of neonatal mouse ovary since it could better support formation of primordial follicles. Yet BMK did not promote follicular activation as well as GCCM prepared with KSR did, and therefore, requires modifications.

Differences in expression of genes related to steroidgenesis in abdominal subcutaneous adipose tissue of pregnant women with and without PCOS; a case control study.

BACKGROUND: It was reported that steroid-related gene expressions in the adipose tissue (AT) of women differ between women affected with polycystic ovary syndrome (PCOS) and non-PCOS. Although association between PCOS in mother and offspring's health is a crucial issue, there are few studies focusing on AT of pregnant women suffering from PCOS. Our objectives were to determine the differences between mRNA expression levels of key steroid-converting enzymes in abdominal subcutaneous AT of pregnant women afflicted with PCOS and non-PCOS. METHODS: Twelve pregnant women with PCOS (case) and thirty six non-PCOS pregnant women (control) (1:3 ratio; age- and BMI-matched) undergoing cesarean section were enrolled for the present study. Expressions of fifteen genes related to steriodogenesis in abdominal subcutaneous AT were investigated using quantitative real-time PCR. RESULTS: No significant differences were detected with respect to age, BMI (prior pregnancy and at delivery day), gestational period and parity among pregnant women with PCOS and non-PCOS. Most of the sex steroid-converting genes except 17ß-Hydroxysteroid dehydrogenases2 (17BHSD2), were highly expressed on the day of delivery in subcutaneous AT. Women with PCOS showed significantly higher mRNA levels of steroidgenic acute regulator (STAR; P < 0.001), cytochrome P450 monooxygenase (CYP11A1; P < 0.05), 17α-hydroxylase (CYP17A1; P < 0.05), and 11ß-Hydroxysteroid dehydrogenase (11BHSD1 and 11BHSD2; P < 0.05). The expression of steroid 21-hydroxylase (CYP21) in non-PCOS was fourfold higher than those of women with PCOS (P < 0.001). There were no significant differences between relative expression of aromatase cytochrome P450 (CYP19A1), 3ß-hydroxysteroid dehydrogenase (3BHSD1 and 3BHSD2), and 17BHSD family (1, 3, 5, 7, and 12) between the two groups. CONCLUSION: The expression levels of genes related to sex steroids metabolism were similar to age-matched and BMI- matched pregnant non-PCOS and pregnant women with PCOS at delivery day. However, the alterations in gene expressions involved in glucocorticoids and mineralocorticoids metabolism were shown. It is necessary to point out that further studies regarding functional activity are required. More attention should be given to AT of pregnant women with PCOS that was previously ignored.

Supplementation of granulosa cells conditioned medium with pyruvate and testosterone could improve early follicular development in cultured 1-day-old mouse ovaries.

In vitro activation of primordial follicles could serve as a safe method to preserve fertility in patients with cancer subjected to ovarian tissue cryopreservation during oncotherapy, however the culture medium for this purpose requires to be optimized. Granulosa cell conditioned medium (GCCM) has been recognized to enhance primordial follicle activation and the present study was conducted to understand whether addition of pyruvate, a combination of insulin, transferrin and selenium (ITS) or testosterone to GCCM could improve its efficiency in this regard. To this end, 1-day-old mouse ovaries were cultured in four different media including CON (control; containing GGCM only), PYR (containing GCCM plus pyruvate), ITS (containing GCCM plus ITS) or TES (containing GCCM plus testosterone) for 11 days. Furthermore, follicular dynamics and gene expression of factors involved in follicular development were assessed using histological examination and RT-PCR, respectively, on days 5 and 11 of culture. Pyruvate decreased follicular activation, but it enhanced the progression of follicles to the primary stage. Moreover, it upregulated Bmp15 and Cx37 (P < 0.05). In the ITS group, activation of follicles was not affected and total number of follicles was reduced by day 11 of culture. Additionally, ITS downregulated Pi3k, Gdf9, Bmp15 and Cx37 (P < 0.05). Although testosterone did not affect primordial follicle activation, it enhanced the development of follicles up to the preantral stage (P < 0.05). Furthermore, testosterone inhibited the expression of Pten but stimulated the expression of Gdf9 and Cx37 (P < 0.05). In conclusion, the present study revealed that inclusion of pyruvate and testosterone into GCCM could enhance the early development of follicles in cultured 1-day-old mouse ovaries.