Effect of carvacrol antioxidant capacity on oocyte maturation and embryo production in cattle.

Carvacrol (C10H14O), an efficient phenolic antioxidant substance for several cell types, may become a useful antioxidant for female germ cells and embryo culture. This study investigates the effects of carvacrol supplementation on bovine oocytes in in vitro maturation (IVM) and embryo production. In total, 1222 cumulus-oocyte complexes were cultured in TCM-199+ alone (control treatment) or supplemented with carvacrol at the concentrations of 3 µM (Carv-3), 12.5 µM (Carv-12.5), or 25 µM (Carv-25). After IVM, the oocytes were subjected to in vitro fertilization and embryo production, and the spent medium post-IVM was used for evaluating the levels of reactive oxygen species and the antioxidant capacity (2,2-diphenyl-1-picryl-hydrazyl-hydrate and 2,2'-azinobis-3-ethyl-benzothiozoline-6-sulphonic acid quantification). A greater (P < 0.05) antioxidant potential was observed in the spent medium of all carvacrol-treated groups compared with the control medium. Moreover, the addition of carvacrol to the maturation medium did not affect (P > 0.05) blastocyst production on days 7 and 10 of culture; however, the total number of cells per blastocyst was reduced (P < 0.05) in two carvacrol-treated groups (Carv-3 and Carv-25). In conclusion, carvacrol demonstrated a high antioxidant capacity in the spent medium after oocyte maturation; however, although embryo production was not affected, in general, carvacrol addition to IVM medium reduced the total number of cells per blastocyst. Therefore, due to the high antioxidant capacity of carvacrol, new experiments are warranted to investigate the beneficial effects of lower concentrations of carvacrol on embryo production in cattle and other species.

In vitro embryo production from early antral follicles of goats fed with a whole full-fat linseed based diet.

The present study aimed to use an in vitro follicle culture (IVFC) biotechnique as a tool to evaluate the influence of whole flaxseed as a feed supplementation in the diet on the in vitro development of caprine early antral follicles (EAFs) and further embryo production. In total, 18 adult goats were homogeneously allocated into two diet groups: Control and Flaxseed. EAFs from both experimental groups (300-400 µm) were isolated and cultured in vitro for 18 days. After IVFC, recovered cumulus-oocyte complexes were submitted to in vitro maturation, and subsequently to IVF and in vitro embryo culture. The endpoints evaluated were follicular growth and morphology, oocyte recovery rate and diameter, sperm penetration, pronuclei formation, embryo development, and estradiol production. The addition of the whole flaxseed in the diet did not affect (P > 0.05) follicular growth and diameter. A higher (P < 0.05) percentage of oocytes ≥ 110 µm was recovered from the flaxseed treatment. However, the sperm penetration rate was higher (P < 0.05) in the control treatment when compared with the flaxseed treatment, but no differences were found regarding the rate of fertilization nor cleaved embryos. In conclusion, dietary flaxseed increased the recovery rate of fully grown oocytes, but it did negatively affect the sperm penetration rate, even though there was no further effect on the cleavage rate.

Application of a dot blot hybridization assay for genotyping Streptococcus uberis from Brazilian dairy herds.

Streptococcus uberis is a major cause of environmental mastitis in many regions, and it is associated with clinical and subclinical infections. Although the main source of infection is the environment, reports of strains with a contagious profile have been described. Dot blot hybridization analysis allows the rapid identification of S. uberis population structures within and between herds, and it helps to identify strain diversity as well as possible clonal lineages that directly affect the control of bovine mastitis caused by this pathogen. The aim of this study was to evaluate the diversity of S. uberis isolates obtained from clinical (n = 22) and subclinical (n = 22) cases of mastitis in dairy herds (n = 13) in Brazil over a period of 12 mo. We submitted 44 S. uberis isolates to dot blot hybridization followed by automatic data analysis. We identified 8 different hybridization patterns using genetic markers associated with virulence factors and taxonomy, indicating diversity of S. uberis within the population and suggesting environmental transmission. However, the evidence of identical dot blot patterns in different mammary quarters from the same animal also suggested local contagious transmission. Of the virulence genes evaluated, we found a high prevalence of the genes sua, pauA, and gapC, highlighting the importance of these virulence factors for the adhesion, invasion, and multiplication of S. uberis in subclinical and clinical intramammary infections.

Effect of dietary crude protein degradability and corn processing on lactation performance and milk protein composition and stability.

The present study aimed to evaluate the effect of crude protein degradability and corn processing on lactation performance, milk protein composition, milk ethanol stability (MES), heat coagulation time (HCT) at 140°C, and the efficiency of N utilization for dairy cows. Twenty Holstein cows with an average of 162 ± 70 d in milk, 666 ± 7 kg of body weight, and 36 ± 7.8 kg/d of milk yield (MY) were distributed in a Latin square design with 5 contemporaneous balanced squares, 4 periods of 21 d, and 4 treatments (factorial arrangement 2 × 2). Treatment factor 1 was corn processing [ground (GC) or steam-flaked corn (SFC)] and factor 2 was crude protein (CP) degradability (high = 10.7% rumen-degradable protein and 5.1% rumen-undegradable protein; low = 9.5% rumen-degradable protein and 6.3% rumen-undegradable protein; dry matter basis). A significant interaction was observed between CP degradability and corn processing on dry matter intake (DMI). When cows were fed GC with low CP degradability, DMI increased by 1.24 kg/d compared with cows fed GC with high CP degradability; however, CP degradability did not change DMI when cows were fed SFC. Similar interactions were observed for MY, HCT, and lactose content. When cows were fed GC diets, high CP degradability reduced MY by 2.3 kg/d, as well as HCT and lactose content, compared with low CP degradability. However, no effect of CP degradability was observed on those variables when cows were fed SFC diets. The SFC diets increased dry matter and starch total-tract digestibility and reduced ß-casein (CN) content (% total milk protein) compared with GC diets. Cows fed low-CP degradability diets had higher glycosylated κ-CN content (% total κ-CN) and MES, as well as milk protein content, 3.5% fat-corrected milk, and efficiency of N for milk production, than cows fed high-CP degradability diets. Therefore, GC and high-CP degradability diets reduced milk production and protein stability. Overall, low CP degradability increased the efficiency of dietary N utilization and MES, probably due to changes in casein micelle composition, as CP degradability or corn processing did not change the milk concentration of ionic calcium. The GC diets increased ß-CN content, which could contribute to reducing HTC when cows were fed GC and high-CP degradability diets.

Anethole improves blastocysts rates together with antioxidant capacity when added during bovine embryo culture rather than in the in vitro maturation medium.

We performed the exposure of bovine oocytes to anethole during in vitro maturation (0 or 300 µg/ml), during in vitro embryo production (0, 30, 300 or 2000 µg/ml), or during both periods to determine the rates of 2-4 cells embryos, blastocysts rates and cells numbers, as well as the production of reactive oxygen species (ROS). Bovine ovaries (n = 240) were collected from a local abattoir after slaughter and cumulus-oocyte complexes (COCs) with homogeneous and non-dark cytoplasm, surrounded by two or more compact layers of cumulus cells, and an intact zona pellucida were selected for in vitro maturatuion (IVM). Mature oocytes were then submitted to in vitro fertilization (IVF) and in vitro embryo production (IVP) in culture medium supplemented or not with different concentrations of anethole, as described above. Although IVM medium supplementation with 300 µg/ml anethole improved the rates of bovine blastocysts formation, we demonstrated that IVP medium supplementation with 30 µg/ml anethole, regardless of IVM medium enrichment, considerably enhanced blastocysts rates. Furthermore, ROS levels were decreased only when anethole was added to the IVP medium without previous IVM medium supplementation.

In vivo and in vitro strategies to support caprine preantral follicle development after ovarian tissue vitrification.

The aim of the present study was to compare fresh and vitrified goat ovarian tissue after autotransplantation and in vitro culture. Adult goats were completely ovariectomised and each ovarian pair was sliced and distributed among six different treatment groups: fresh control, fresh transplant, fresh culture, vitrified control, vitrified transplant and vitrified culture. Follicular morphology, development, growth, density, revascularisation and hormone production were evaluated in all groups. Three antral follicles (two in the fresh transplant and one in the vitrified transplant groups) were observed on the surface of the graft 90 days after transplantation. The percentage of morphologically normal follicles was similar in the fresh control, fresh transplant and vitrified transplant groups. The percentage of developing (transition, primary and secondary) follicles was higher after in vitro culture of fresh or vitrified tissue. Transplantation resulted in a lower follicle density. Serum oestradiol concentrations remained constant during the entire transplantation period. In contrast, progesterone production decreased significantly. Expression of CD31 mRNA was lower in fresh culture. In conclusion, restoration of goat ovarian function can be successfully achieved following transplantation of both fresh and vitrified goat ovarian tissue. However, transplantation induced higher follicle loss than in vitro culture.

Melatonin reduces apoptotic cells, SOD2 and HSPB1 and improves the in vitro production and quality of bovine blastocysts.

Effects of adding different concentrations of melatonin (10-7 , 10-9 and 10-11  M) to maturation (Experiment 1; Control, IVM + 10-7 , IVM + 10-9 , IVM + 10-11 ) and culture media (Experiment 2; Control, IVC + 10-7 , IVC + 10-9 , IVC + 10-11 ) were evaluated on in vitro bovine embryonic development. The optimal concentration of melatonin (10-9  M) from Experiments 1-2 was tested in both maturation and/or culture media of Experiment 3 (Control, IVM + 10-9 , IVC + 10-9 , IVM/IVC + 10-9 ). In Experiment 1, maturated oocytes from Control and IVM + 10-9 treatments showed increased glutathione content, mitochondrial membrane potential and percentage of Grade I blastocysts (40.6% and 43%, respectively). In Experiment 2, an increase in the percentage of Grade I blastocysts was detected in IVC + 10-7 (43.5%; 56.7%) and IVC + 10-9 (47.4%; 57.4%). Moreover, a lower number and percentage of apoptotic cells in blastocysts were observed in the IVC + 10-9 group compared to Control (3.8 ± 0.6; 3.6% versus 6.1 ± 0.6; 5.3%). In Experiment 3, the IVC + 10-9 treatment increased percentage of Grade I blastocysts with a lower number of apoptotic cells compared to IVM/IVC + 10-9 group (52.6%; 3.0 ± 0.5 versus 46.0%; 5.4 ± 1.0). The IVC + 10-9 treatment also had a higher mRNA expression of antioxidant gene (SOD2) compared to the Control, as well as the heat shock protein (HSPB1) compared to the IVM + 10-9 . Reactive oxygen species production was greater in the IVM/IVC + 10-9 treatment group. In conclusion, the 10-9  M concentration of melatonin and the in vitro production phase in which it is used directly affected embryonic development and quality.

Ovarian fragment sizes affect viability and morphology of preantral follicles during storage at 4°C.

The method of transportation and the conditions imposed on the ovarian tissue are pivotal aspects for the success of ovarian tissue cryopreservation (OTC). The aim of this study was to evaluate the effect of the size of the ovarian tissue (e.g. whole ovary, biopsy size and transplant size) during different times of storage (0, 6, 12 and 24 h) on the structural integrity of equine ovarian tissue transported at 4°C. Eighteen pairs of ovaries from young mares (<10 years old) were harvested in a slaughterhouse and processed to simulate the fragment sizes (biopsy and transplant size groups) or kept intact (whole ovary group) and stored at 4°C for up to 24 h in α-MEM-enriched solution. The effect of the size of the ovarian tissue was observed on the morphology of preantral follicles, stromal cell density, DNA fragmentation and mitochondrial membrane potential. The results showed that (i) biopsy size fragments had more morphologically normal preantral follicles after 24 h of storage at 4°C; (ii) mitochondrial membrane potential was the lowest during each storage time when the whole ovary was used; (iii) DNA fragmentation rate in the ovarian cells of all sizes of fragments increased as storage was prolonged and (iv) transplant size fragments had increased stromal cell density during storage at cool temperature. In conclusion, the biopsy size fragment was the best to preserve follicle morphology for long storage (24 h); however, transportation/storage should be prior determined according to the distance (time of transportation) between patient and reproduction centers/clinics.

Preantral follicle density in ovarian biopsy fragments and effects of mare age.

The aims of the present study were to: (1) evaluate preantral follicle density in ovarian biopsy fragments within and among mares; (2) assess the effects of mare age on the density and quality of preantral follicles; and (3) determine the minimum number of ovarian fragments and histological sections needed to estimate equine follicle density using a mathematical model. The ovarian biopsy pick-up method was used in three groups of mares separated according to age (5-6, 7-10 and 11-16 years). Overall, 336 preantral follicles were recorded with a mean follicle density of 3.7 follicles per cm2. Follicle density differed (P<0.05) among animals, ovarian fragments from the same animal, histological sections and age groups. More (P<0.05) normal follicles were observed in the 5-6 years (97%) than the 11-16 years (84%) age group. Monte Carlo simulations showed a higher probability (90%; P<0.05) of detecting follicle density using two experimental designs with 65 histological sections and three to four ovarian fragments. In summary, equine follicle density differed among animals and within ovarian fragments from the same animal, and follicle density and morphology were negatively affected by aging. Moreover, three to four ovarian fragments with 65 histological sections were required to accurately estimate follicle density in equine ovarian biopsy fragments.

Alpha lipoic acid controls degeneration and ensures follicular development in ovine ovarian tissue cultured in vitro.

This study aims to evaluate the effects of adding alpha lipoic acid (ALA) to the in vitro ovarian tissue culture medium, either fresh or after vitrification/warming. For this purpose, 10 ovaries from five adult sheep were used. Each pair of ovaries gave rise to 16 fragments and were randomly distributed into two groups: fresh (n = 8) and vitrified (n = 8). Two fresh fragments were fixed immediately and considered the control, while another six were cultured in vitro for 14 days in the absence; presence of a constant (100 µM/0-14 day) or dynamic (50 µM/day 0-7 and 100 µM/day 8-14) concentration of ALA. As for the vitrified fragments, two were fixed and the other six were cultured in vitro under the same conditions described for the fresh group. All the fragments were subjected to morphological evaluation, follicular development and stromal density (classical histology), DNA fragmentation (TUNEL), senescence (Sudan Black), fibrosis (Masson's Trichome), and endoplasmic reticulum stress (immunofluorescence). Measurements of the antioxidant capacity against the free radicals 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,2-diphenyl-1-picryl-hydrazyl-hydrate (DPPH) and estradiol (E2) levels in the culture medium was performed. The results showed that in the absence of ALA, in vitro culture of vitrified ovarian fragments showed a significant reduction (P < 0.05) in follicular morphology and increased the presence of senescence and tissue fibrosis (P < 0.05). Dynamic ALA maintained E2 levels unchanged (P > 0.05) until the end of vitrified ovarian tissue culture and controlled the levels of ABTS and DPPH radicals in fresh or vitrified cultures. Therefore, it is concluded that ALA should be added to the vitrified ovarian tissue in vitro culture medium to reduce the damage that leads to loss of ovarian function. To ensure steroidogenesis during in vitro culture, ALA should be added dynamically (different concentrations throughout culture).

Transcriptional downregulation of ABC transporters is related to follicular degeneration after vitrification and in vitro culture of ovine ovarian tissue.

ATP-binding cassette (ABC) transporters perform multiple functions in reproductive tissues. During ovarian tissue vitrification, the plasma membrane has important functions in the influx or efflux of water, and substances such as cryoprotectants and channel proteins that are required in this process. Thus, the present study aimed to verify the relative abundance of mRNA transcript of ABC transporters ABCB1, ABCG2, and MRP2 after vitrification and in vitro culture (IVC) of ovine ovarian tissue. For this study, the ovarian cortex fragments were proportioned into four groups as fresh control, vitrified control, fresh culture, and vitrified culture groups. After vitrification and in vitro culture, the ovarian tissue was evaluated using morphological procedures. Further, relative abundance of ABCB1, ABCG2, and MRP2 transporter mRNA transcripts in the ovarian cortex subjected to aforementioned treatment conditions were evaluated using qPCR. Our results showed a negative association between degenerated follicles and mRNA transcript abundances of ABCB1 and ABCG2. In addition, the percentage of growing follicles in the ovine ovarian cortex after vitrification was similar to that of the fresh control tissue without in vitro culture. The in vitro culture of fresh and vitrified tissue however, showed a significant decrease in the percentage of growing follicles. To the best of our knowledge, we believe that our data for the first time has studied the relative abundances of ABCB1 and ABCG2 mRNA transcripts in the ovine ovarian cortex. In addition, alterations of these protein channels may be indicative of a deleterious effect of osmotic stress on follicular survival during vitrification. Furthermore, these effects were detectable only after the IVC of the ovarian tissues. Nonetheless, further studies are required to investigate the functions of ABC transporters in ovine folliculogenesis, especially after in vitro culture of ovarian tissue.

Eugenol Improves Follicular Survival and Development During in vitro Culture of Goat Ovarian Tissue.

This study evaluated the effects of different concentrations (10, 20, or 40 µM) of eugenol (EUG 10, EUG 20, or EUG 40), ascorbic acid (50 µg/mL; AA) or anethole (300 µg/mL; ANE 300) on the in-vitro survival and development of goat preantral follicles and oxidative stress in the cultured ovarian tissue. Ovarian fragments from five goats were cultured for 1 or 7 days in Alpha Minimum Essential Medium (α-MEM+) supplemented or not with AA, ANE 300, EUG 10, EUG 20 or EUG 40. On day 7 of culture, when compared to MEM, the addition of EUG 40 had increased the rate of follicular development, as observed by a decrease in the proportion of primordial follicles alongside with an increase in the rate of normally developing follicles. Furthermore, EUG 40 significantly increased both follicular and oocyte diameters. Subsequently, ovarian fragments from three goats were cultured for 1 or 7 days in α-MEM+ supplemented or not with AA, ANE 300 or EUG 40. All tested antioxidants, except ANE 300, were able to significantly decrease the levels of reactive oxygen species in the ovarian tissue, but EUG 40 could most efficiently neutralize free radicals. All ovarian tissues cultured in the presence of antioxidants, especially EUG 40, presented a significant decrease in H3K4me3 labeling, indicating a silencing of genes that play a role in the inhibition of follicular activation and apoptosis induction. When compared to cultured control tissues, both EUG 40 and ANE 300 significantly increased the intensity of calreticulin labeling in growing follicles. The mRNA relative expression of ERP29 and KDM3A was significantly increased when the culture medium was supplemented with EUG 40, indicating a response to ER stress experienced during culture. In conclusion, EUG 40 improved in-vitro follicle survival, activation and development and decreased ROS production, ER stress and histone lysine methylation in goat ovarian tissue.

In vitro- and in vivo-derived early antral follicles have comparable in vitro follicular growth and oocyte maturation rates in goats.

Recent in vitro follicle culture (IVFC) studies in caprine have yielded lower maturation rates using late preantral follicles compared to early antral follicles. Thus, research focusing on developing stage-specific customized culture systems able to improve the efficiency of IVFC for late preantral follicles are warranted. This study aimed to compare the morphometric features, estradiol production, and gene expression between early antral caprine follicles produced in vitro and in vivo. In vitro-derived early antral follicles were produced after a 6-day in vitro culture of late preantral follicles, while in vivo-derived early antral follicles were yielded immediately after isolation from the ovaries; antral follicles were, thereafter, cultured for 18 days. In vitro-derived antral follicles were cultured either in a medium developed for preantral follicles (PF medium) or in a medium developed for antral follicles (AF medium). In vivo-derived early antral follicles, on the other hand, were cultured in AF medium (Control treatment). Results demonstrated that in vitro-derived antral follicles cultured in PF medium produced higher estradiol concentration, and m-RNA expression for matrix metalloproteinase-9 (MMP-9), and insulin receptor when compared to both in vitro- and in vivo-derived antral follicles cultured in AF medium. Remarkably, in vitro-derived antral follicles cultured in PF medium had similar MII and oocytes ≥110 µm rates compared with in vivo-derived antral follicles (Control treatment). In conclusion, when cultured in a single and appropriate medium (i.e., PF medium), in vitro-derived early antral follicles had comparable oocyte maturation rates to the in vivo-derived early antral follicles.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst.

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 µM (EU-10), 20 µM (EU-20), or 40 µM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 µM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 µM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 µM EU for in vitro bovine embryo production.

Impact of ethanol and heat stress-dependent effect of ultra-diluted Arnica montana 6 cH on in vitro embryo production in cattle.

This study evaluated the effect of adding ultra-diluted and dynamized Arnica montana 6 cH, and its vehicle (0.3% ethanol) to the in vitro maturation (IVM) medium, in the absence (experiment 1) or presence (experiment 2) of heat stress (HS), on bovine oocyte maturation and in vitro embryo production (IVEP). In experiment 1 (n = 902 cumulus oocyte complexes, COCs), the treatments were 1) IVM medium (Control treatment), 2) IVM medium + 0.3% ethanol, and 3) IVM medium + Arnica montana 6 cH. In experiment 2 (n = 1064 COCs), the treatments were 1) IVM medium without HS, 2) IVM medium under HS, 3) IVM medium + ethanol under HS, and 4) IVM medium + Arnica montana under HS. In the absence of HS (experiment 1), the addition of Arnica montana to the IVM medium had a deleterious effect on the IVEP (cleavage and blastocyst rates) and the total cell number/blastocysts. On the other hand, ethanol (0.3%) increased IVEP in relation to the Control and Arnica montana treatments. However, in the presence of HS during IVM (experiment 2), the addition of ethanol or Arnica montana increased IVEP when compared to the HS treatment alone, and the Arnica montana treatment resulted in greater total cell number/blastocysts compared to the other treatments. In conclusion, this study showed for the first time that the negative or positive effect of Arnica montana 6 cH on IVEP depends on the culture condition (i.e., absence or presence of HS during IVM). On the other hand, ethanol showed beneficial and consistent results on IVEP regardless of exposure to HS.

Heterotopic autotransplantation of equine ovarian tissue using intramuscular versus subvulvar grafting sites: Preliminary results.

Ovarian tissue transplantation (OTT) is a technique well established and successfully applied in humans using mainly orthotopic or heterotopic transplantation sites. In livestock, OTT is still in its infancy and, therefore, different aspects of the technique, including the efficiency of different heterotopic OTT sites as well as the potential effect of age (i.e., young vs. old mares) in the ovarian graft quality, need to be investigated. The present study investigated the efficacy of the intramuscular (IM) or the novel subvulvar mucosa (SV) heterotopic autotransplantation sites to maintain the survivability of the grafts for 3 and 7 days post-OTT. Ovarian biopsy fragments were obtained in vivo and distributed to the following treatments: Fresh control group (ovarian fragments immediately fixed), SV-3, IM-3, SV-7, and IM-7. During and after graft harvesting, the macroscopic characteristics of the grafts (i.e., adherence, morphology, and bleeding) were scored, and the percentages of morphologically normal and developing preantral follicles as well as the follicular and stromal cell densities of the grafts were evaluated. The results were that similar (P > 0.05) macroscopic scores were observed between both transplantation sites 7 days post-OTT, with positive correlations (P < 0.01) found among adherence, morphology, and bleeding of the grafts. A lower (P < 0.05) percentage of morphologically normal follicles was found 7 days post-OTT in the SV site (82%) compared with the Fresh control group (99%) and IM site (95%); however, the percentages of developing follicles were similar (P > 0.05) between both transplantation sites 7 days post-OTT (30-43%). Although similar (P > 0.05) follicular densities were found in both transplantation sites in young and old mares at 3 and 7 days post-OTT, large individual variation in the follicular depletion rate was observed regardless of transplantation site. The Fresh control group and SV-7 treatments had higher (P < 0.05) stromal cell densities in young and old mares compared with both IM-7 treatments. When comparing transplant sites between young and old mares, the follicular density in old mares and the stromal cell density in young mares were greater (P < 0.05) in the SV than in the IM site. In conclusion, even though the transplantation sites differentially affected some end points, overall comparable findings of the OTT technique using both heterotopic autotransplantation sites (i.e., IM and SV) for equine ovarian tissue were observed.

Harvesting, processing, and evaluation of in vitro-manipulated equine preantral follicles: A review.

The mammalian ovary is responsible for essential stages of folliculogenesis and hormonal production, regulating the female physiological functions during the menstrual/estrous cycles. The mare has been considered an attractive model for comparative studies due to the striking similarities shared with women regarding in vivo and in vitro folliculogenesis. The ovarian follicular population in horses contains a large number of oocytes enclosed in preantral follicles that are yet to be explored. Therefore, the in vitro manipulation of equine preantral follicles aims to avoid the process of atresia and promote the development of follicles with competent oocytes. In this regard, after ovarian tissue harvesting, the use of appropriate processing techniques, as well as suitable approaches to evaluating equine preantral follicles and ovarian tissue, are necessary. Although high-quality equine ovarian tissue can be obtained from several sources, some critical aspects, such as the age of the animals, ovarian cyclicity, reproductive phase, and the types of ovarian structures, should be considered. Therefore, this review will focus on providing an update on the most current advances concerning the critical factors able to influence equine preantral follicle quality and quantity. Also, the in vivo strategies used to harvest equine ovarian tissue, the approaches to manipulating ovarian tissue post-harvesting, the techniques for processing ovarian tissue, and the classical approaches used to evaluate preantral follicles will be discussed.

Supportive techniques to investigate in vitro culture and cryopreservation efficiencies of equine ovarian tissue: A review.

During the reproductive lifespan of a female, only a limited quantity of oocytes are naturally ovulated; therefore, the mammalian ovary possesses a substantial population of preantral follicles available to be handled and explored in vitro. Hence, the manipulation of preantral follicles enclosed in ovarian tissue aims to recover a considerable population of oocytes of high-value animals for potential application in profitable assisted reproductive technologies (ARTs). For this purpose, the technique of preantral follicle in vitro culture (IVC) has been the most common research tool, achieving extraordinary results with offspring production in the mouse model. Although promising outcomes have been generated in livestock animals after IVC of preantral follicles, the quantity and quality of embryo production with those oocytes are still poor. In recent years, the mare has become an additional model for IVC studies due to remarkable similarities with women and livestock animals regarding in vivo and in vitro ovarian folliculogenesis. For a successful IVC system, several factors should be carefully considered to provide an optimum culture environment able to support the viability and growth of preantral follicles enclosed in ovarian tissue. The cryopreservation of the ovarian tissue is another important in vitro manipulation technique that has been used to preserve the reproductive potential in humans and, in the future, may be used in highly valuable domestic animals or endangered species. Several improvements in cryopreservation protocols are necessary to support the utilization of ovarian tissue of different species in follow-up ARTs (e.g., ovarian fragment transplantation). This review aims to provide an update on the most current advances regarding supportive in vitro techniques used in equids to evaluate and manipulate preantral follicles and ovarian tissue, as well as methodological approaches used during IVC and cryopreservation techniques.

The subtle balance of insulin and thyroxine on survival and development of in vitro cultured caprine preantral follicles enclosed in ovarian tissue.

Numerous studies have reported the importance of thyroid hormones on the development of later preantral and antral follicles, but their interactions with other hormones and effects in regulating early preantral follicle growth remain unclear. Here we investigated the in vitro effects of thyroxine combined with insulin on caprine preantral follicle survival and development. Sliced ovarian tissues were cultured for 1 or 7 days using 10 ng/mL (low) or 10 µg/mL (high) insulin in the presence of thyroxine at 0, 0.5, 1 or 2 µg/mL. Post-culture, we evaluated the follicular survival and development, assessed the expression of apoptotic-related genes (Bcl2/Bax) and receptors of insulin and thyroid hormones, and quantified the estradiol and reactive oxygen species (ROS) production levels. Follicular survival in low-insulin culture conditions was enhanced by the presence of 0.5 µg/mL thyroxine (P < 0.05) as compared to the thyroxine-free medium but remained similar to non-cultured control in the presence of 2 µg/mL (P > 0.05). Significantly higher ROS production was measured from Day 1 to Day 7 in low-insulin culture media containing 0.5 or 2 µg/mL thyroxine (P < 0.05). When compared to high insulin level, the presence of thyroxine in low insulin culture conditions yielded higher stromal cell density (P < 0.05), increased estradiol production on Day 1, and higher Bcl2/Bax ratio on Day 7. Cultures with high levels of both insulin and thyroxine led to follicles and oocytes with larger diameters (P < 0.05). The RNA transcript levels of insulin and thyroid receptors were reduced in the presence of high insulin cultures when compared to controls (non-cultured). In conclusion, the combination of low concentrations of insulin and thyroxine better maintained follicle survival, while high levels ensured better follicular development.

Cilostamide affects in a concentration and exposure time-dependent manner the viability and the kinetics of in vitro maturation of caprine and bovine oocytes.

This study investigated: 1) the kinetics of oocyte chromatin configuration during in vitro maturation (IVM) of caprine and bovine oocytes; and 2) the effect of in vitro pre-maturation (IVPM) with cilostamide with or without association of the follicular wall (FW) on the same parameters. In experiment I, cumulus-oocyte complexes (COCs) were cultured in vitro in a standard maturation medium for 6, 12, 18 or 30 h. For experiment II, the COCs were cultured for 30 h, either in a standard IVM medium or in IVPM containing cilostamide (10 or 20 µM) and FW alone or in combination, for 6 or 12 h before the onset of maturation. The MII rate was similar (P > .05) between 18 and 30 h of maturation, both of which were higher (P < .05) than 6 and 12 h IVM in both species (Experiment I). Contrary to caprine, all IVPM treatments presented a higher (P < .05) percentage of bovine oocytes arrested at the GV stage than the control treatment after 6 h of culture. The percentage of MII oocytes after 30 h (IVPM+IVM) of culture in bovine oocytes treated with 10 µM cilostamide associated with FW and FW alone cultured for 6 h presented MII percentages similar to the control. However, in caprine, these treatments significantly reduced the percentages of MII in relation to the control treatment (Experiment II). In conclusion, the combination of concentration-exposure time to cilostamide during IVPM delayed meiotic progression in bovine after 6 and 12 h of culture. However, overall the culture period (IVPM+IVM) influenced the oocyte chromatin configuration and kinetics in both species.