Sheep Ovaries Research Articles

Ovarian follicular dynamics and plasma steroid concentrations are not significantly different in ewes given intravaginal sponges containing either 20 or 40 mg of fluorogestone acetate

Although various progestagens are often used to induce and synchronize estrus and ovulation in ruminants, concerns regarding residues are the impetus to develop alternative approaches, including reduced doses of progestagens. Therefore, the objective was to determine whether ovarian function was affected by halving the dose of fluorogestone acetate in intravaginal sponges for synchronizing ovulation in sheep during the physiologic breeding season. Twenty Manchega ewes, 4–6-year-old, were randomly allocated to receive an intravaginal sponge containing either 20 mg (P20, n = 10) or 40 mg of fluorogestone acetate (P40, n = 10). Cloprostenol (125 μg) was given at sponge insertion, and all sponges were removed after 6 d. Ovarian follicular dynamics (monitored by daily ultrasonography) and other aspects of ovarian function did not differ significantly between the two groups. Ovulatory follicles (OF) grew at a similar growth rate ( r = 0.62; P < 0.001), with comparable initial and maximum diameters (4.2 ± 0.4 to 6.0 ± 0.3 mm in P20 vs. 4.6 ± 0.6 to 5.7 ± 0.2 mm in P40, mean ± S.E.M.). Plasma estradiol concentrations (determined once daily) increased linearly during the 72 h interval after sponge removal (1.3 ± 0.1 to 3.3 ± 0.1 pg/mL for P20, P < 0.005 and 1.4 ± 0.1 to 3.1 ± 0.2 pg/mL for P40, P < 0.005). Ten days after sponge removal, ovulation rates (1.2 ± 0.2 for P20 and 1.4 ± 0.3 for P40), and plasma progesterone concentrations (3.8 ± 0.35 ng/mL for P20 and 3.9 ± 0.38 ng/mL for P40) were similar. In conclusion, reducing the dose of fluorogestone acetate from 40 to 20 mg did not affect significantly ovarian follicular dynamics or other aspects of ovarian function.

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2–3 h after collection. Cumulus–oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24 h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4–6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4–6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.

Cryopreservation of Whole Murine and Porcine Livers

Preservation of vascularized organs, such as the liver, is limited to 24 h before destructive processes disqualify them for transplantation. This narrow window of opportunity prevents the performance of optimal pathogen screening and matching tests and possibly results in the need for retransplantation. Numerous problems are associated with freezing and thawing a whole liver while preserving its viability upon thawing, including complicated geometry, poor heat transfer, release of latent heat, and the difficulty of generating a uniform cooling rate. On the basis of our past success with sheep ovaries, we have now applied our novel freezing technique to a larger solid organ, the liver. Whole rat and pig livers were frozen and thawed using directional solidification apparatus, and viability of these livers was tested by means of integrity and functionality in vitro and in auxiliary liver transplantation. The thawed rat and porcine livers were intact and demonstrated >80% viability. Histology revealed normal architecture. Bile production and blood flow following auxiliary transplantation were normal as well. Our encouraging results in applying this novel cryopreservation technique in rat and pig livers suggest that this method may enable better human organ donor-recipient matching in the future.

The Role of BMP Signalling Pathway in the Regulation of Ovarian Follicle Development.

Bone morphogenetic proteins (BMPs) regulation of ovarian function has been investigated in several animal models, demonstrating their role in primordial germ cell formation, primordial-to-primary follicle recruitment, follicle development, steroidogenesis and ovulation. However, the mechanism by which BMPs regulate individual processes of ovarian function is not well understood. The aim of this study was to investigate the effect of the BMP receptor mutation in Booroola sheep on primordial follicle formation and rate of recruitment in comparison to that in normal merino sheep in vivo. Whole ovaries from Booroola and merino sheep at birth, 1.5 years and 5 years of age were obtained by bilateral ovariectomy and processed for the quantification of primordial and primary follicles using computerised stereological methods (Stereo Investigator Software Version 7, MicroBrightField, Inc.) and JMP 6 statistical package (SAS Institute Inc, 2005). Regardless of animal strain, the overall absolute number of primordial and primary follicles per ovary of newborn sheep was considerably higher than in the 1.5 and 5-year old ewes. Log transformed data analyses revealed that the total number of primordial follicles in newborn Booroola sheep was significantly (P=0.0065) lower than in merino of the same age. At 1.5 years a trend showing a considerable reversal in the number of primordial follicles was observed in Booroola ewes, which then significantly (p<0.0036) increased at 5 years of age. The rate of primordial follicle recruitment was substantially lower in Booroola ewes compared with that in merino, as reflected by the difference in the absolute number of primordial follicles remaining at 1.5 and 5 years of age. Immediately after birth, the mean total number of primordial follicles in Booroola strain was only 36% of that present in the merino strain. This ratio became 207% and 367% at 1.5 and 5 years, respectively. The ratios of primordial follicle consumption in Booroola ewes were 51% and 66% up to the age of 1.5 and 5 years respectively compared to 92% and 97% of primordial follicles have been used in merino ewes. On the other hand, the mean numbers of primary follicles per ovary in newborn animals was similar in both sheep strains. However, at 1.5 years, Booroola ewes possess more primary follicles than merino, and the absolute number of primary follicles present at 1.5 years was equal to 26% of the number present at birth, compared with only 8% of primary follicles present in merino ewes. At 5 years of age the primary follicles were not counted in either animal strain. These findings suggest that attenuation of the intraovarian signaling pathway of BMPs may in fact be a successful means of rationalizing follicle consumption, preventing unnecessary loss of follicles from the initial follicle pool, hence increasing reproductive age and fertility.

Maternal Dietary Restriction Affects Follicle Stimulating Hormone (FSH) Receptor (R) Protein Expression in Fetal Ovaries of Sheep.

Maternal Dietary Restriction Affects Follicle Stimulating Hormone (FSH) Receptor (R) Protein Expression in Fetal Ovaries of Sheep.

Difficulties improving ovarian functional recovery by microvascular transplantation and whole ovary vitrification

To evaluate recovery of endocrine function and fertility after transplantation and vitrification of whole ovaries. Animal study. Lyon Veterinary School, France. Ewes. In group 1 (n = 5), the left ovary was removed with its vascular pedicle and was transplanted onto the contralateral pedicle. In group 2 (n = 5), the left ovary with its pedicle was cryopreserved after a vitrification procedure. After thawing, transplantation was performed by microvascular anastomosis to the contralateral ovarian pedicle. Median ischemia time, progesterone levels, histologic examination. Successful microsurgical transplantation was performed in both groups. The median ischemia time was statistically significantly longer in group 2 (287 minutes, range: 226 to 349] versus 129 minutes [range: 125 to 130]) in group 1. In group 1, four sheep recovered spontaneous ovarian endocrine function about 2.5 (range: 2.00 to 3.75) months after transplantation. Two ewes gave healthy live births at 12 and 25 months, respectively, after transplantation. In group 2, one ewe recovered ovarian endocrine function 6 months after transplantation. However, histologic evaluation showed a follicular survival rate of 6% in group 1, and total follicle loss in group 2. Autograft of whole sheep ovaries with microvascular anastomosis seems technically feasible but resulted in a very poor follicle survival rate (6%), in spite of endocrine function recovery and birth of two lambs. Attempts at cryopreservation with vitrification resulted in no follicle survival at all.

In vitro maturation of sheep oocytes in different concentrations of mare serum

The aim of the study was to determine the optimum concentration of the mare serum (MS) for sheep in vitro oocyte maturation. Sheep ovaries were collected from a local abattoir and transported within 1 h to the laboratory in a warm saline solution (30 – 35oC), supplemented with 100 IU penicillin G and 100
g streptomycin sulfate/ml. Cumulus-oocyte complexes (COC’s) were obtained by slicing of follicles, washed in TCM-199 modification with NaHCO3 and supplemented with 50
g/ml gentamycin, and 0.25 mM sodium pyruvate without any serum supplementation. The COC’s were randomly divided into four groups. Group 1 (n = 105) COC’s were fresh control and cultured in TCM-199 medium without serum supplementation. Group 2 (n = 108) COC’s were washed five times and cultured in TCM-199 medium supplemented with 10% MS. Group 3 (n = 112) COC’s were washed five times and cultured in TCM-199 medium supplemented with 15% MS. Group 4 (n = 114) COC’s were washed five times and cultured in TCM-199 medium supplemented with 20% MS. After 38 - 42 h of IVM, oocytes were denuded with the aid of 0.1% hyaluronidase and passing them through a fine pipette, fixed for 24 – 48 h in a mixture of acetic acid and alcohol (1:3) at room temperature, stained for 10 min with 1% (w/v) orcein in 45% acetic acid and examined for the evidence of different stages of maturation. Significantly higher (p those without serum supplementation. However, no significant difference was observed between the 10, 15 and 20% serum supplemented group.

Effect of exogenous melatonin on the ovary, the embryo and the establishment of pregnancy in sheep

Administration of melatonin to advance the breeding season in sheep has been widely used, since this hormone conveys the photoperiodic signal to the reproductive neuroendocrine axis. An increased lambing percentage has been reported following such treatment during anoestrus, which could be mediated through a higher rate of embryonic survival, either by an improvement in luteal function or by a reduction in the antiluteolytic mechanisms. The aim of this article is to review the body of knowledge on the effect of melatonin on the ovine ovary, the embryo and the establishment of pregnancy. Some studies using synchronized ewes have found that melatonin treatments during anoestrus do not improve ovulation rate by modifying the timing of follicle emergence, but increasing the number of ovulatory follicles by decreasing the atresia of medium and large follicles. On the other hand, the addition of melatonin to the in vitro maturation medium does not improve oocyte maturation rate in oocytes from sheep ovaries recovered either in anoestrus or in the breeding season. However, a luteotrophic effect of melatonin at either short or medium term has been reported. We have recently observed that melatonin implants tend to improve the survival of embryos collected from ewes after superovulation in anoestrus. More specifically, melatonin induced a significant reduction of the number and rate of non-viable (degenerate and retarded) embryos. Preliminary data from our laboratory suggest that the uterine sensitivity to progesterone – in terms of progesterone receptor expression – of superovulated ewes could be reduced by melatonin treatment. It can be concluded that the success of exogenous melatonin as a means to improve lamb production of sheep is due, at least in part, to an improvement of luteal support and embryonic survival.

209 PRODUCTION OF IN VITRO-FERTILIZED INTERSPECIES BLASTOCYSTS BETWEEN SHEEP OOCYTES AND GOAT SPERMATOZOA

In rodents, chimeric blastocysts produced by combining embryonic cells of 2 different species have been used to investigate cell lineage and cell interaction during development. Interspecific chimerism offers new approaches to the study of reproductive incompatibilities between species. The aim of the present study was to produce interspecies embryos between sheep oocytes and goat spermatozoa through in vitro fertilization. Sheep ovaries were collected from a nearby abattoir and transported to the laboratory in 0.9% normal sterile saline containing antibiotics (50 μg mL–1 of gentamicin sulfate) at 30 to 35°C. Oocytes were aspirated by the puncturing method in a medium consisting of TCM-199 and 3 mg mL–1 of BSA. Only A and B grade COC with 3 or more layers of cumulus cells with homogeneous ooplasm were taken for maturation. The oocytes were washed 4 to 5 times in maturation medium containing TCM-199 (HEPES modified), 10 μg mL–1 of LH, 5 μg mL–1 of FSH, 1 μg mL–1 of estradiol-17β, 50 μg mL–1 of sodium pyruvate, 5.5 mg mL–1 of glucose, 3.5 μg mL–1 of L-glutamine, 50 μg mL–1 of gentamicin, 3 mg mL–1 of BSA, and 10% EGS (heat-inactivated goat serum). The COC (15 to 20 oocytes) were placed in 100-μL droplets of maturation medium, covered with paraffin oil in a 35-mm Petri dish, and incubated in a CO2 incubator (5% CO2 in air) with maximum humidity at 38.5°C for 24 h. Fresh semen was collected from a proven buck. The semen was washed at 300g 2 times in sperm-TALP (Parrish et al. 1986) medium to remove the seminal plasma and incubated with fert-TALP medium containing sperm-TALP supplemented with 50 μg mL–1 of heparin and 3 mg mL–1 of BSA for 1.5 h for capacitation. The matured sheep oocytes with expanded cumulus cells were coincubated with capacitated buck spermatozoa at a concentration of 2 × 106 sperm mL–1 for 10 h in 5% CO2 in air with maximum humidity at 38.5°C. The presumptive zygotes were then cultured in embryo development medium containing TCM-199 (HEPES modified), 0.03 mg mL–1 of sodium pyruvate, 0.1 mg mL–1 of L-glutamine, 0.05 mg mL–1 of gentamicin, 10 μL mL–1 of essential amino acids, 5 μL mL–1 of nonessential amino acids, 10 mg mL–1 of BSA (fraction V), 10% fetal calf serum, and 50 mm cysteamine along with sheep oviductal cells for further development. The cleavage was recorded at 36 to 48 h postinsemination, and morula- and blastocyst-stage embryos were obtained on Day 5 and Day 7, respectively. The cleavage percentage was found to be 58.6%. Among the cleaved embryos, 43% reached the morula stage, and among morula, 31% reached the blastocyst stage. We concluded that interspecies embryos between sheep and goat can be produced successfully in vitro up to the blastocyst stage. Table 1. In vitro production of different stages of interspecies (sheep × goat) embryos

Short- and Long-Term Effects of Unilateral Ovariectomy in Sheep: Causative Mechanisms1

The mechanisms of ovulatory compensation following unilateral ovariectomy (ULO) are still not understood. In the present study, we investigated the short- and long-term effects of ULO in sheep using transrectal ovarian ultrasonography and hormone estimations made during the estrous cycle in which surgery was done, the estrous cycle 2 mo after surgery, and the 17-day period during the subsequent anestrus. The ULOs were done when a follicle in the first follicular wave of the cycle reached a diameter > or =5 mm, leaving at least one corpus luteum and one ovulatory-sized follicle in the remaining ovary. Ovulation rate per ewe was 50% higher in the ULO ewes compared with the control ewes at the end of the cycle during which surgery was performed, but it did not differ between groups at the end of the cycle, 2 mo later. This compensation of ovulation rate in ULO ewes was due to ovulation of follicles from the penultimate follicular wave in addition to those from the final wave of the cycle. Ovulation from multiple follicular waves appeared to be due to a prolongation of the static phase of the largest follicle of the penultimate wave of the cycle. Interestingly, the length of the static phase of waves was prolonged in ULO ewes compared with control ewes in every instance where the length of the static phase could be determined. Changes in follicular dynamics due to ULO were not associated with alterations in FSH and LH secretion. In conclusion, ovulatory compensation in ULO sheep involves ovulation from multiple follicular waves due to the lengthened static phase of ovulatory-sized follicles. These altered antral follicular dynamics do not appear to be FSH or LH dependent. Further studies are required to examine the potential role of the nervous system in the enhancement of the life span of the ovulatory-sized follicles leading to ovulatory compensation by the unpaired ovary in ULO sheep.

Construction of SMART cDNA Library of Sheep Ovary and Identification of Candidate Gene by Homologous Cloning

The cDNA library of an ovary from Small Tail Han sheep before estrus was constructed by switching mechanism at 5 end of RNA transcript (SMART) approach. This library had a plaque liter of 1 × 10 9 pfu mL −1 and a 96% recombinant ratio of which the fragment length of inserted average cDNA sequences was 1.0 kb. Based on bioinformatics analysis of the sequences, we obtained 338 expressed sequence tags (ESTs) from 380 cDNA clones which indicated 191 contigs. These contigs consist of 89 unmatched ESTs, 9 homologous known genes in sheep, and 93 homologous sequences in species of mouse, bovine, and human beings, including 19 sequences expressed in the ovary or follicle and 14 unknown sequences. Several candidate genes associated with sheep reproduction trait such as epidermal growth factor (EGF), estrogen receptor (ESR), Inhibin, follicle stimulating hormone receptor (FSHR), prostaglandin (PG), and transforming growth factor-β (TGF-β) were identified and the homologous were cloned from this library, which will contribute to compile expression profiles and find the major genes of prolificacy of Small Tail Han sheep.

Ovarian survival 6 years after whole organ cryopreservation and transplantation

It was previously reported (Arav et. al.2005) that ovarian function continued for up to 3 years after whole organ cryopreservation. Now we report the longest functional term survival (6 years) of autotransplanted frozen/thawed whole sheep ovaries. Descriptive research to perfect whole ovary cryopreservation/thawing procedures. Nine month old Assaf sheep were used for in vivo experiments (n = 3). Briefly, under general anesthesia one of the ovaries was excised leaving the contralateral in place. The ovarian artery was perfused with 10 ml of cold (4°C) UW supplemented with 10% DMSO for 3 minutes and then inserted into a glass freezing tube for cryopreservation. Unidirectional solidification freezing methodology reduced the temperature to –30°C at the very slow speed of 0.01 mm/sec, resulting in a cooling rate of −0.3°C/min after which the tubes were plunged into liquid nitrogen. Thawing was performed by placing the tubes into a 37°C water bath. Cryoprotectants were rinsed out from the thawed ovary via the ovarian artery using 10 ml Hepes-Talp medium supplemented with 0.5 M sucrose and 10 iu/ml heparin. Ovarian auto transplantation was performed by microscopic end-to-end vascular (artery and vein) reanastomoses to the contralateral ovarian vascular pedicle using 10–0 sutures at the same time that the intact ovary was removed. 6 years following transplantation sheep were sacrificed and the ovaries were excised, visually inspected, fixed in Bouin's liquid and embedded in paraffin for histology. Progesterone levels were measured in transplanted sheep up to 6 years post transplantation, twice per week indicating normal ovulation. Transplanted ovaries were of normal size and shape showing no necrotic lesions and one ovary clearly showed a recent corpus luteum. A total of 36 antral follicles were observed by transillumination and after sectioning, there were 4 GV's oocytes that matured in vitro to MII stage. Histological evaluation using Eosin & Hematoxylene revealed normal tissue architecture including follicles at various developmental stages and intact blood vessels. This report documents the longest term survival of whole ovary cryopreserved and then re-transplanted. Cryopreservation of whole ovaries using directional freezing and microvascular anastomoses is a promising method for preserving long-term reproductive capacity and endocrine function.

Effects of vitrification on functional recovery of ovarian tissues

Effects of vitrification on functional recovery of ovarian tissues

Towards whole sheep ovary cryopreservation

Cryopreservation of ovarian tissue aims to assist young women who require treatments that may lead to sterility or infertility. Cryopreservation procedures should therefore be as simple and efficient as possible. This study investigates rapid cooling outcomes for whole sheep ovaries. Ovaries were perfused with VS4 via the ovarian artery, and cooled by quenching in liquid nitrogen in less than a minute (estimated cooling rate above 300 °C/min till the vitreous transition temperature). The ovaries were rewarmed in two stages: slow warming (12–16 °C/min from −196 to −133 °C) in liquid nitrogen vapour, followed by rapid thawing in a 45 °C water bath at about 200 °C/min. DSC measurements showed that under these cryopreservation conditions VS4 would vitrify, but that VS4 perfused ovarian cortex fragments did not vitrify, but formed ice (around 18.4%). Immediately following rewarming, a dye exclusion test indicated that 61.4 ± 2.2% of small follicles were viable while histological analysis showed that 48 ± 3.8% of the primordial follicles were normal. It remains to be clarified whether follicle survival rates will increase if conditions allowing complete tissue vitrification were used.

Kisspeptin Synchronizes Preovulatory Surges in Cyclical Ewes and Causes Ovulation in Seasonally Acyclic Ewes

We determined whether kisspeptin could be used to manipulate the gonadotropin axis and ovulation in sheep. First, a series of experiments was performed to determine the gonadotropic responses to different modes and doses of kisspeptin administration during the anestrous season using estradiol-treated ovariectomized ewes. We found that: 1) injections (iv) of doses as low as 6 nmol human C-terminal Kiss1 decapeptide elevate plasma LH and FSH levels, 2) murine C-terminal Kiss1 decapeptide was equipotent to human C-terminal Kiss1 decapeptide in terms of the release of LH or FSH, and 3) constant iv infusion of kisspeptin induced a sustained release of LH and FSH over a number of hours. During the breeding season and in progesterone-synchronized cyclical ewes, constant iv infusion of murine C-terminal Kiss1 decapeptide-10 (0.48 mumol/h over 8 h) was administered 30 h after withdrawal of a progesterone priming period, and surge responses in LH occurred within 2 h. Thus, the treatment synchronized preovulatory LH surges, whereas the surges in vehicle-infused controls were later and more widely dispersed. During the anestrous season, we conducted experiments to determine whether kisspeptin treatment could cause ovulation. Infusion (iv) of 12.4 nmol/h kisspeptin for either 30 or 48 h caused ovulation in more than 80% of kisspeptin-treated animals, whereas less than 20% of control animals ovulated. Our results indicate that systemic delivery of kisspeptin provides new strategies for the manipulation of the gonadotropin secretion and can cause ovulation in noncyclical females.

Comparative Histomorphological Study of Ovary and Ovarian Follicles in Iranian Lori-Bakhtiari Sheep and Native Goat

In this project, for measuring and comparing dimensions of ovary and ovarian follicles between Lori-Bakthiari sheep and native goat, eighty ovaries of sheep and goat, aged 1-2 years, from Shahrekord abattoir were collected. After weighing the ovaries, some factors such as: length, width and thickness of left and right ovaries by caliper device were measured. For histological studies, after tissue preparation and staining with H and E, diameter of follicles, thickness of follicular layers (granulosa, theca interna and theca externa) by micrometry methods were measured. By analyzing data with t-test and comparing them between sheep and goat we concluded that, there were significant difference between weight and dimensions of ovaries in sheep and goat. These parameters in goats were larger than sheep. Microscopical studies revealed that, the mean thickness of theca externa of graafian follicle in left ovary of sheep was more than right. The mean diameter of oocyte in secondary follicle of goat in left ovary was more than right. Also in goat, the number of layers of cumulus oophorus around corona radiata was more than sheep.

Cryopreservation of Sheep Primordial Follicles

The aim of this study was to evaluate the efficiency of 1 M dimethylsulphoxide (DMSO), ethylene glycol (EG), propylene glycol (PROH) and glycerol (GLY) to cryopreserve primordial follicles. The first evaluation was performed soon after cryopreservation and the second evaluation after 4 days of in vitro culture, using the cryoprotectants that allowed the higher results (higher follicular survival rate) after cryopreservation. The results after follicular isolation (control) and cryopreservation using 1 M DMSO, EG, PROH and GLY showed that the mean number (+/- SEM) of live follicles per millilitre was 3204 (100%) +/- 319.27, 2798 (87%) +/- 239.14, 2492 (78%) +/- 345.8, 448 (14%) +/- 46.3 and 208 (7%) +/- 75.26, respectively. Higher follicular survival was reported when DMSO and EG were used. Control follicles and follicles cryopreserved with these two cryoprotectants were cultured and the percentage of follicular survival was 55% (control), 42% (EG) and 34% (DMSO). Similar results were found between control and follicles cryopreserved with EG. In conclusion, 1 M EG is the most effective cryoprotectant to preserve primordial follicles isolated from ovaries of sheep.

54. Physical investigations aiming to achieve the cryopreservation of the whole sheep ovary by vitrification

54. Physical investigations aiming to achieve the cryopreservation of the whole sheep ovary by vitrification

The Effects of Immunizing Sheep with Different BMP15 or GDF9 Peptide Sequences on Ovarian Follicular Activity and Ovulation Rate1

The aims of these studies were to determine the abilities of antisera against different regions of ovine bone morphogenetic protein 15 (BMP15) and growth differentiation factor 9 (GDF9) to inhibit ovarian follicular activity, estrus (mating), and ovulation in sheep. The 9-15-mer peptides were conjugated to keyhole limpet hemocyanin (KLH) and used to generate antibodies against the flexible N-terminal regions of the mature protein as well as against regions in which dimerization of the protein or interaction with a type 1 BMP or a type 2 TGFB or BMP receptor was predicted to occur. Ewes (n = 10 per treatment group) were vaccinated with KLH or the KLH-BMP15 (n = 9 different peptides) or KLH-GDF9 (n = 10) peptides in Freund adjuvant at five consecutive monthly intervals. Overall, antisera generated against peptides that corresponded to amino acid residues 1-15 of the N-terminus of the BMP15 or GDF9 mature protein or GDF9 amino acid residues 21-34 were the most potent at inhibiting ovulation following primary and single booster vaccination. Several other BMP15 (8/9) or GDF9 (6/10) treatment groups, but not KLH alone, also produced significant reductions in the numbers of animals that ovulated, although 2, 3 or 4 booster vaccinations were required. Anovulation was commonly associated with the inhibition of normal ovarian follicular development and anestrus. The in vitro neutralization studies with IgG from the BMP15 or GDF9 immunized ewes showed that the mean inhibition of BMP15 plus GDF9 stimulation of (3)H-thymidine uptake by rat granulosa cells was approximately 70% for animals without corpora lutea (CL), whereas for animals with one to three CL or more than three CL, the inhibition was 24%-33% or 27%-42%, respectively. In summary, these data suggest that reagents that block the biological actions of BMP15 or GDF9 at their N-termini have potential as contraceptives or sterilizing agents.

Cryopreservation of the ovary by vitrification as an alternative to slow-cooling protocols

To study the thermal properties of a cryoprotectant solution, called VS4, and of VS4-impregnated whole sheep ovaries with pedicle. Physical and experimental animal study. Academic research environment. Five- to 6-month-old ewes. Thermal properties on cooling of a cryoprotectant solution called VS4 were measured by differential scanning calorimetry. VS4 contains 2.75 mol/L dimethyl sulfoxide, 2.76 mol/L formamide, and 1.97 mol/L propylene glycol. Whole sheep ovaries were collected at the slaughterhouse and prepared for a vitrification procedure. Cortex and vessels underwent histologic examination before and after vitrification, and the thermal properties of VS4-impregnated ovarian cortex were then studied. Critical cooling rates (V(ccr)), vitreous transition temperature (Tg), end-of-melting temperature (Tm), follicle viability assessment by trypan blue test, and histologic examination of ovary and vessel structure. The critical cooling rate (V(ccr)) of VS4 solution was estimated to be 14.3 +/- 1.1 degrees C/min. Its vitreous transition temperature (Tg) was -125.2 +/- 0.2 degrees C, and its end-of-melting temperature (Tm) -34.3 +/- 0.1 degrees C. Following our vitrification procedure, immediate follicle viability was 61.4% +/- 2.2%. The percentage of normal primordial follicles remaining after vitrification was 48% +/- 3.8%. The V(ccr) of VS4-impregnated cortex could not be determined because of the quantity of ice forming as of the top programmed cooling rate (-300 degrees C/min). The mean ovarian cortex cooling rate actually attained experimentally during vitrification was -342.9 +/- 49.6 degrees C/min. Vitrification of entire organs, such as ovaries, is a great challenge in cryobiology and reproductive medicine. Physical studies seem indispensable for progress with this technique. Thus, our ovarian perfusion procedure needs improving to enhance ovarian cortex impregnation and bring down the V(ccr) rate in both tissue and VS4 solution.