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.

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.

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.

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.

The mule (Equus mulus) as a recipient of horse (Equus caballus) embryos: Comparative aspects of early pregnancy with mares.

The aim of this study was to compare the embryonic and early fetal development of horse embryos between recipient mules and mares from day 10-60 of pregnancy, in addition to hormonal (eCG and progesterone), ovarian, and uterine characteristics for approximately 4 months. Embryo donor mares (n = 5) and two groups of recipients (acyclic mules, n = 7; cyclic mares, n = 7) were used. Donor mares were monitored daily by transrectal ultrasonography and inseminated using fresh semen. Cyclic recipient mares were synchronized with the donor's ovulation using PGF2α and deslorelin acetate. Mules were prepared for the embryo transfers with estrogen and progestagen. Embryo collection and transfer were performed 8 days after ovulation of the donor mares. Pregnancy diagnosis with ultrasonography began 1 day after embryo transfer. After pregnancy confirmation, the recipient mules received long-acting progesterone once weekly for at least 120 days. The first day of detection (day 10) of an embryonic vesicle (EV) was similar between mules and mares. A period of extensive intrauterine mobility of the embryonic vesicle was observed similarly in mules and mares from days 10-17. The day of fixation of the EV in mules tended to be 1-day earlier than in mares; however, the diameter and growth rate of the EV did not differ between the two species. The embryo proper was first detected at day 20, and the crown-rump, width, and diameter were similar between the two recipient types. The heartbeat and allantoic sac tended to be detected 1 day later in mules than in mares, while the umbilical cord was first observed around day 40 in both species. Besides the expected differences found in ovarian aspects and eCG production, similar endometrial diameter, uterine tone and echotexture, and progesterone levels were seen between the two types of recipients. In conclusion, striking ultrasound similarities in equine embryo and fetal development, and uterine characteristics were seen between mules and mares used as recipients of horse embryos.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles.

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 µg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 µg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Reproductive system development in male and female horse embryos and fetuses: Gonadal hyperplasia revisited.

In horses, pregnancy is characterized by high levels of maternal estrogens that are produced largely by the interstitial tissue inside the gonads of the offspring, associated with a physiological gonadal hyperplasia, that is uncommon in other species. However, a detailed structural-functional understanding of the early stages of gonadal development and hyperplasia has remained elusive in horse pregnancy because of the lack of substantial data. The goal of this study was to describe the genital organs' development in 19 early horse embryos and fetuses (days 20-140 of gestation) of both sexes by means of anatomy, histology, stereology, and immunohistochemistry, with a specific focus on gonadal hyperplasia and interstitial tissue development. Gonadal hyperplasia with similar amounts of interstitial cells was observed in both sexes, but only during the early stage of development (days 40-90). Surprisingly, a higher degree of hyperplasia, characterized by larger amounts of interstitial cell-rich areas, was seen in fetal ovaries from 90 days of gestation onwards. Another novel aspect was that parallel to the hyperplasia of the interstitial cells, a much more precocious and pronounced differentiation of germinal cells was seen in the ovary, characterized by an earlier peak and decrease of DAZL and OCT protein immune markers. In conclusion, a reduced degree of hyperplasia and interstitial tissue in the fetal testis after 90 days of gestation suggests the existence of a more efficient mechanism regarding the synthesis of estrogen precursors as a structural or physiological difference between both fetal sexes, which warrants further investigation.

Alpha lipoic acid (ALA) effects on developmental competence of equine preantral follicles in short-term culture.

The effect of different concentrations of alpha lipoic acid (ALA) on the development and morphology of preantral follicles, as well as the proliferative activity of granulosa cells, was assessed after short-term culture. Ovaries (n = 5) of five seasonal anestrous mares were harvested in a local abattoir. At the laboratory, nine ovarian fragments (5 × 5 × 1 mm) from each animal were used. One fragment was immediately fixed in Bouin and subjected to histological and immunohistochemistry (proliferating cell nuclear antigen, PCNA) analyses (noncultured group; D0 = day 0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ plus ALA (50, 100, or 250 µM). After culture, fragments were subjected to histology and PCNA analyses. After two days of culture, ALA 50 and ALA 100 had the greatest (P < 0.05) percentage of normal primordial follicles (97.2 and 95.1%, respectively), when compared to other groups, and did not differ (P > 0.05) from the fresh noncultured control group. Furthermore, the total percentage of normal follicles was greater (P < 0.05) in the ALA 50 and ALA 100 than in the MEM-D2 group. After six days of culture, the highest (P < 0.05) proliferative activity of granulosa cells in developing follicles was observed for the groups MEM+ (92.9%), ALA 50 (100%), and ALA 100 (96.4%). In conclusion, the results of this study demonstrated that (1) ALA 50 and ALA 100 preserved the morphological integrity of equine primordial follicles for up two days of culture, and (2) granulosa cells of developing follicles enclosed in ovarian tissue and cultured for up to six days in MEM+ with or without ALA were highly stained by PCNA.

Anethole reduces oxidative stress and improves in vitro survival and activation of primordial follicles

Primordial follicles, the main source of oocytes in the ovary, are essential for the maintenance of fertility throughout the reproductive lifespan. To the best of our knowledge, there are no reports describing the effect of anethole on this important ovarian follicle population. The aim of the study was to investigate the effect of different anethole concentrations on the in vitro culture of caprine preantral follicles enclosed in ovarian tissue. Randomized ovarian fragments were fixed immediately (non-cultured treatment) or distributed into five treatments: α-MEM+ (cultured control), α-MEM+ supplemented with ascorbic acid at 50 μg/mL (AA), and anethole at 30 (AN30), 300 (AN300), or 2000 µg/mL (AN2000), for 1 or 7 days. After 7 days of culture, a significantly higher percentage of morphologically normal follicles was observed when anethole at 2000 μg/mL was used. For both culture times, a greater percentage of growing follicles was observed with the AN30 treatment compared to AA and AN2000 treatments. Anethole at 30 and 2000 µg/mL concentrations at days 1 and 7 of culture resulted in significantly larger follicular diameter than in the cultured control treatment. Anethole at 30 µg/mL concentration at day 7 showed significantly greater oocyte diameter than the other treatments, except when compared to the AN2000 treatment. At day 7 of culture, levels of reactive oxygen species (ROS) were significantly lower in the AN30 treatment than the other treatments. In conclusion, supplementation of culture medium with anethole improves survival and early follicle development at different concentrations in the caprine species.

Role of EGF on in situ culture of equine preantral follicles and metabolomics profile.

The effects of epidermal growth factor (EGF) concentrations (0, 10, 50, and 100ng/ml) on in vitro culture (IVC) of equine preantral follicles were evaluated using histology, estradiol and reactive oxygen species (ROS) production and metabolomics. After IVC, the percentage of normal follicles was lower (P<0.05) for all treatments when compared to non-cultured control. EGF 50ng/ml treatment had more (P<0.05) normal follicles at Day 7 of culture when compared with EGF 0 and 100ng/ml. EGF 50ng/ml had more (P<0.05) developing follicles than the 0ng/ml and 10ng/ml EGF treatments. Follicular and oocyte diameters were greater (P<0.05) with EGF 50ng/ml than the other cultured treatments, but similar (P>0.05) to the non-cultured control. From Day 1 to Day 7 estradiol production increased (P<0.05) in all EGF treatments. EGF 50ng/ml was the only treatment that maintained ROS production through IVC. Metabolomics profiles of the spent media indicated that eleven ions from variable influence in the projection (VIP) scores were higher represented in the EGF 50ng/ml treatment. In conclusion, EGF 50ng/ml treatment maintained follicle survival and ROS production, and promoted activation of cultured equine preantral follicles enclosed in ovarian tissue.

Novel prospects for evaluation of follicle wall blood flow using color-Dopplerultrasonography

The goal of this study was to develop anobjective method for evaluation of ovarian follicle wallblood flow in cattle. Two subjective methods were used:(I) real-time ultrasound evaluations performed by oneoperator in the barn and (II) video clip evaluationsperformed by four operators in the laboratory. Thefollowing objective methods evaluated in the laboratorywere used for comparison: (I) percentage of follicle wallcircumference under blood flow (WUF) and (II) pixelarea of color-Doppler signals. Cows (n = 21) weresubmitted to a synchronization protocol, follicles ≥7 mmwere measured, and blood flow was evaluated every 12 huntil ovulation using color-Doppler ultrasonography. Nodifference (P > 0.05) was observed among laboratoryoperators from day 2 of training onwards. Therefore,an average score of all operators was used forcomparisons among different methods. Both subjectiveand objective methods of evaluation showed anincrease (P < 0.0001) in follicle blood flow over time.Higher (P < 0.001) correlations were obtained betweenWUF and subjective laboratory evaluation thanbetween WUF and pixel area or WUF and subjectivebarn data. Higher (P < 0.0003) correlation coefficientswere observed for WUF than for the pixel area whencompared with the barn (r = 0.70 vs. r = 0.42) orlaboratory (r = 0.84 vs. r = 0.62) data. Subjectiveevaluations at the laboratory and barn producedstronger correlations with WUF (P < 0.0008) than withpixel area (P < 0.01). In conclusion, WUF is aneffective and reliable method for objective evaluationof follicle wall blood flow in cows.(AU)

Novel prospects for evaluation of follicle wall blood flow using color-Dopplerultrasonography

The goal of this study was to develop anobjective method for evaluation of ovarian follicle wallblood flow in cattle. Two subjective methods were used:(I) real-time ultrasound evaluations performed by oneoperator in the barn and (II) video clip evaluationsperformed by four operators in the laboratory. Thefollowing objective methods evaluated in the laboratorywere used for comparison: (I) percentage of follicle wallcircumference under blood flow (WUF) and (II) pixelarea of color-Doppler signals. Cows (n = 21) weresubmitted to a synchronization protocol, follicles ≥7 mmwere measured, and blood flow was evaluated every 12 huntil ovulation using color-Doppler ultrasonography. Nodifference (P > 0.05) was observed among laboratoryoperators from day 2 of training onwards. Therefore,an average score of all operators was used forcomparisons among different methods. Both subjectiveand objective methods of evaluation showed anincrease (P < 0.0001) in follicle blood flow over time.Higher (P < 0.001) correlations were obtained betweenWUF and subjective laboratory evaluation thanbetween WUF and pixel area or WUF and subjectivebarn data. Higher (P < 0.0003) correlation coefficientswere observed for WUF than for the pixel area whencompared with the barn (r = 0.70 vs. r = 0.42) orlaboratory (r = 0.84 vs. r = 0.62) data. Subjectiveevaluations at the laboratory and barn producedstronger correlations with WUF (P < 0.0008) than withpixel area (P < 0.01). In conclusion, WUF is aneffective and reliable method for objective evaluationof follicle wall blood flow in cows.

Relationships between follicle and corpus luteum diameter, blood flow, and progesterone production in beef cows and heifers: preliminary results

The aim of the present study in beef cattle was to investigate potential differences in follicle size and follicle wall-blood flow between cows and heifers and to compare follicle wall-blood flow between smaller and larger follicles. Cows and heifers were treated with a synchronization protocol and follicles and CLs were measured and evaluated for blood flow. Pregnancy diagnosis was performed on day 50 of the protocol. Cows had larger (P < 0.008) follicles than heifers. Cows, heifers, and pregnant and non-pregnant cows did not differ (P > 0.05) in CL diameter, CL blood flow, and plasma progesterone concentrations. Moderate correlations between follicle diameter and follicle blood flow were observed for cows (r = 0.51; P < 0.002) and heifers (r = 0.61; P < 0.0001). Pregnant cows tended (P < 0.1) to have larger follicles between 12 to 60 h before ovulation, and had larger (P < 0.05) follicles than non-pregnant cows at hour 24 before ovulation and at hour 12 before maximum values. Pregnant cows had greater (P < 0.05) follicle blood flow than non-pregnant cows at hours −36 and −24 before maximum values. Follicle blood flow was greater (P < 0.002) in the large follicles compared with the small follicles, and tended (P < 0.06) to be greater than in medium follicles. Moderate to strong correlations were found between follicle blood flow and diameter of small (r = 0.59; P < 0.002), medium (r = 0.50; P < 0.02), and large (r = 0.71; P < 0.0001) follicles. Pregnancy rates were similar (P > 0.05) among all follicle diameter categories. In conclusion, synchronized beef cows and pregnant cows had larger follicles and greater blood flow than heifers and non-pregnant cows, and follicle wall blood flow was closely associated with increasing follicle diameter.(AU)

Relationships between follicle and corpus luteum diameter, blood flow, and progesterone production in beef cows and heifers: preliminary results

The aim of the present study in beef cattle was to investigate potential differences in follicle size and follicle wall-blood flow between cows and heifers and to compare follicle wall-blood flow between smaller and larger follicles. Cows and heifers were treated with a synchronization protocol and follicles and CLs were measured and evaluated for blood flow. Pregnancy diagnosis was performed on day 50 of the protocol. Cows had larger (P 0.05) in CL diameter, CL blood flow, and plasma progesterone concentrations. Moderate correlations between follicle diameter and follicle blood flow were observed for cows (r = 0.51; P 0.05) among all follicle diameter categories. In conclusion, synchronized beef cows and pregnant cows had larger follicles and greater blood flow than heifers and non-pregnant cows, and follicle wall blood flow was closely associated with increasing follicle diameter.

FSH supplementation to culture medium is beneficial for activation and survival of preantral follicles enclosed in equine ovarian tissue.

This study investigated the effect of adding different concentrations of bovine recombinant follicle-stimulating hormone on the IVC of equine preantral follicles enclosed in ovarian tissue fragments. Randomized ovarian fragments were fixed immediately (fresh noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0, 10, 50, and 100 ng/mL FSH and subsequently analyzed by classical histology. Culture media collected on Day 1 or Day 7 and were analyzed for steroids (estradiol and progesterone) and reactive oxygen species (ROS). After Day 1 and Day 7 of culture, 50-ng/mL FSH treatment had a greater (P < 0.05) percentage of morphologically normal follicles when compared to the other groups, except the 10-ng/mL FSH treatment at Day 1 of culture. The percentage of developing follicles (transition, primary, and secondary), and follicular and oocyte diameters were higher (P < 0.05) in the 50-ng/mL FSH treatment compared to the other groups after Day 7 of culture. Furthermore, estradiol secretion and ROS production were maintained (P > 0.05) throughout the culture in the 50-ng/mL FSH treatment. In conclusion, the addition of 50 ng/mL of FSH promoted activation of primordial follicles to developing follicles, improved survival of preantral follicles, and maintained estradiol and ROS production of equine ovarian tissue after 7 days of culture.

Insulin improves in vitro survival of equine preantral follicles enclosed in ovarian tissue and reduces reactive oxygen species production after culture.

This study investigated the effect of insulin concentration on the in vitro culture of equine preantral follicles enclosed in ovarian tissue. Ovarian tissue samples were immediately fixed (noncultured control) or cultured for 1 or 7 days in α-MEM(+) supplemented with 0 ng/mL, 10 ng/mL, or 10 µg/mL insulin. Ovarian tissues were processed and analyzed by classical histology. Culture medium samples were collected after 1 and 7 days of culture for steroid and reactive oxygen species (ROS) analyses. The percentage of morphologically normal follicles was greater (P < 0.001) in insulin-treated groups after 1 day of culture; likewise, more (P < 0.02) normal follicles were observed after 7 days of culture in medium supplemented with 10-ng/mL insulin. Furthermore, an increase (P < 0.01) in developing (transition, primary, and secondary) follicles between Days 1 and 7 of culture was observed only with the 10-ng/mL insulin treatment. ROS production after 1 or 7 days of culture was lower (P < 0.0001) in medium with 10-ng/mL insulin than the other treatments. Ovarian tissues containing preantral follicles were able to produce estradiol and progesterone after 1 and 7 days of culture; however, treatments did not differ in steroid production. In conclusion, the use of a physiological concentration (10 ng/mL) of insulin rather than the previously reported concentration (10 µg/mL) for in vitro culture of equine preantral follicles improved follicular survival and growth and lowered oxidative stress. Results from this study shed light on new perspectives for producing an appropriate medium to improve equine preantral follicle in vitro survival and growth.

Number and density of equine preantral follicles in different ovarian histological section thicknesses.

Regardless of species, advances in preantral follicle culture and cryopreservation and transplant of ovarian tissue techniques are dependent on the number and density of preantral follicles in the ovary. This study tested the effect of different histological section thicknesses on number, classification, and density of equine preantral follicles. An ovarian fragment was obtained from 5- to 10-year-old mares (n = 14) after slaughter, and each fragment was submitted to three histological section thickness treatments: 3, 5, and 7 µm. The area (cm(2)) of each ovarian fragment was measured, and the sections were evaluated by light microscopy. The percentage of morphologically normal follicles (89%) was similar (P > 0.05) among primordial, transitional, and primary follicles and also among histological section thicknesses. A greater (P < 0.05) number of preantral follicles per histological section were seen in the 7-µm (8.0 ± 2.2) than that in the 3-µm (3.4 ± 0.7) treatment. Furthermore, a linear regression analysis reported that the number of preantral follicles increased (P < 0.05) when a thicker section treatment was used. However, no association (P > 0.05) between follicular density and treatment was observed. The mean number of preantral follicles per fragment (45.3 ± 18.8) and the follicular density (3.0 ± 0.5 follicles per cm(2)) were different (P < 0.05) among mares. In conclusion, this study on equine preantral follicles reported that (1) a 7-µm histological section thickness might be recommended because it allowed identification of a greater number of preantral follicles per sample, (2) a large individual variation in follicle population and density was detected regardless of histological section thickness, and (3) mares have a low number and density of preantral follicles when compared with those reported for other species.

Effects of a-MEM and TCM-199 culture media and epidermal growth factor on survival and growth of goat and sheep preantral follicles cultured in vitro

The aim of this study was to evaluate the effect of culture media (Alpha Minimum Essential Medium, α-MEM; and Tissue Culture Medium-199, TCM-199) in the absence or presence of Epidermal Growth Factor (EGF) on an in vitro culture of goat and sheep preantral follicles enclosed in ovarian tissue. The fragments of ovarian cortex from both species were immediately analyzed after collection (non-cultured control group) or cultured for 1 or 7 days in α-MEM+ or TCM-199+in the absence or presence of EGF (10 ng/ml). Before and after the culture, the fragments of ovarian cortex were analyzed by classical histology and fluorescence microscopy. After 1 day of culture, all treatments decreased the percentage of morphologically normal follicles when compared to non-cultured control in both species (P < 0.05). In fluorescence microscopy, viable sheep follicles were observed to decrease in all treatments after 7 days of culture when compared to non-cultured controls. However, in goats, the culture with TCM-199+maintained follicle viability after 7 days of culture, similar to fresh tissue (P > 0.05). Regarding follicle activation, an increase in the percentage of growing follicles was observed in all treatments after 7 days of culture when compared to the control group in both species. However, in sheep, after 7 days, only the treatments α-MEM+/EGF and TCM-199+showed larger(P < 0.05) oocytes than the control group. In conclusion, the TCM-199+ Preserved goat preantral follicle viability after in vitro culture. Furthermore, the media α-MEM+/EGF and TCM-199+ increased the oocyte diameter after 7 days of culture in sheep. Therefore, it isrecommended to use TCM-199+ In the culture of preantral follicles in both species.(AU)

Effects of a-MEM and TCM-199 culture media and epidermal growth factor on survival and growth of goat and sheep preantral follicles cultured in vitro

The aim of this study was to evaluate the effect of culture media (Alpha Minimum Essential Medium, α-MEM; and Tissue Culture Medium-199, TCM-199) in the absence or presence of Epidermal Growth Factor (EGF) on an in vitro culture of goat and sheep preantral follicles enclosed in ovarian tissue. The fragments of ovarian cortex from both species were immediately analyzed after collection (non-cultured control group) or cultured for 1 or 7 days in α-MEM+ or TCM-199+in the absence or presence of EGF (10 ng/ml). Before and after the culture, the fragments of ovarian cortex were analyzed by classical histology and fluorescence microscopy. After 1 day of culture, all treatments decreased the percentage of morphologically normal follicles when compared to non-cultured control in both species (P 0.05). Regarding follicle activation, an increase in the percentage of growing follicles was observed in all treatments after 7 days of culture when compared to the control group in both species. However, in sheep, after 7 days, only the treatments α-MEM+/EGF and TCM-199+showed larger(P < 0.05) oocytes than the control group. In conclusion, the TCM-199+ Preserved goat preantral follicle viability after in vitro culture. Furthermore, the media α-MEM+/EGF and TCM-199+ increased the oocyte diameter after 7 days of culture in sheep. Therefore, it isrecommended to use TCM-199+ In the culture of preantral follicles in both species.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.