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).

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.

Effects of FSH addition to an enriched medium containing insulin and EGF after long-term culture on functionality of equine ovarian biopsy tissue.

The effect of FSH supplementation on an enriched cultured medium containing insulin (10 ng/mL) and EGF (50 ng/mL) was investigated on in vitro culture of equine ovarian biopsy tissue. Ovarian tissue fragments were collected from mares (n = 10) and distributed in the following treatments: noncultured control, cultured control, and cultured + FSH. Both treated groups were cultured for 7 or 15 days. The end points evaluated were: follicular morphology, estradiol levels in the culture medium, fluorescence intensity for TUNEL, EGFR and Ki-67 detection, and gene expression of GDF-9, BMP-15, and Cyclin-D2 in the ovarian tissue. After seven days of culture, medium supplemented with FSH had a similar (P > 0.05) percentage of morphologically normal follicles compared to the noncultured control group. Estradiol levels increased (P < 0.05) from Day 7 to Day 15 of culture for both treated groups. No difference (P > 0.05) was observed for TUNEL and EGFR intensity between the noncultured control group and the treated groups after 15 days of culture. Ki-67 intensity did not differ (P > 0.05) between treated groups after 15 days of culture, but decreased (P < 0.05) when compared with the noncultured control group. Similar (P > 0.05) mRNA expression for GDF-9, BMP-15, and Cyclin-D2 was observed among all treatments after 15 days of culture. In conclusion, an enriched medium supplemented or not with FSH was able to maintain the functionality of equine ovarian biopsy tissue after a long-term in vitro culture.

Comparative analysis of laparoscopic and ultrasound-guided biopsy methods for gene expression analysis in transgenic goats.

The present study aimed to compare laparoscopic (LP) and ultrasound-guided (US) biopsy methods to obtain either liver or splenic tissue samples for ectopic gene expression analysis in transgenic goats. Tissue samples were collected from human granulocyte colony stimulating factor (hG-CSF)-transgenic bucks and submitted to real-time PCR for the endogenous genes (Sp1, Baff, and Gapdh) and the transgene (hG-CSF). Both LP and US biopsy methods were successful in obtaining liver and splenic samples that could be analyzed by PCR (i.e., sufficient sample sizes and RNA yield were obtained). Although the number of attempts made to obtain the tissue samples was similar (P > 0.05), LP procedures took considerably longer than the US method (P = 0.03). Finally, transgene transcripts were not detected in spleen or liver samples. Thus, for the phenotypic characterization of a transgenic goat line, investigation of ectopic gene expression can be made successfully by LP or US biopsy, avoiding the traditional approach of euthanasia.

Expression of CD44 in sheep oocytes and preimplantation embryos.

The CD44 family belongs to a larger group of hyaluronic acid-binding proteins and plays important roles in oocyte maturation, fertilization and preimplantational embryo development. We analyzed the CD44 receptor in sheep oocytes and embryos. Immature oocytes (N = 66) were obtained from a local abattoir; mature oocytes (N = 35) and embryos (N = 41) were obtained by laparotomy from adult hair ewes submitted to ovarian stimulation treatment. The CD44 mRNA was detected by hemi-nested PCR, after reverse transcription, while proteins were located by indirect immunofluorescence, using anti-human CD44 monoclonal antibody. Human lymphocytes and immature bovine oocytes were used as positive and negative controls, respectively. Assessment of the oocyte nuclear stages as well as classification of the embryonic development stage were made with Hoechst 33342 staining. Indirect immunofluorescence detected CD44 expression on the surface of mature oocytes and embryos; immature oocytes did not take up the stain. These findings were supported by the RT-PCR data, which showed no mRNA templates for CD44, even after two consecutive amplifications, in material from immature oocytes and cumulus cells. The CD44 amplicons were detected after a second hemi-nested PCR in mature oocytes and embryos. The finding of CD44 in mature oocytes and preimplantational embryos could reflect the expression profile of hyaluronic acid during terminal folliculogenesis and preimplantational embryo development in sheep.

Mensurações ultrassonográficas da cisterna da glândula mamária de caprino transgênico / Ultrasound measurements of the mammary gland of transgenic hormone-induced lactating goat

Milk production of transgenic does was evaluated by ultrasound measurements of the mammary gland. Two Canindé goats, which were nine months of age were used in the trial, one non-transgenic or other transgenic for hG-CSF. For hormone-induced lactation, animals were given estradiol (0.25mg/kg, IM), progesterone (0.75mg/kg, IM), and prednisolone (0.4mg/kg, IM). Ultrasonographic exams were carried out during milking, using a Falcon 100 ultrasound equipment with a 5MHz convex probe and were performed by the same operator. The results were expressed as mean±standard error. The maximum greater length and shorter length of the cistern were respectively 5.14cm and 1.36cm for the transgenic animal and 7.28cm and 2.25cm for non-transgenic, which is consistent with the maximum milk volume produced. The relationship between the average area of cisterns and milk yield was expressed as a linear correlation curve, with a correlation coefficient significantly positive for both transgenic (Y=-1.1314+10.8538*x; r=0.97) and non-transgenic (Y=-21.7551+18.3634*x; r=0.97) animals. In conclusion, the ultrasound is a practice and appropriate technique to evaluate the cisterns in ruminant udders in transgenic animal.

Goat oocyte production by standard or one-shot FSH treatments and quantitative analysis of transcripts for EGF ligands and its receptor after in vitro maturation.

Hormonal ovarian stimulation may affect the success of embryo production by regulating transcripts in recovered cumulus-oocyte complexes (COCs). Here, in parallel to morphological classification and in vitro maturation (IVM) rate analysis, we investigated the expression of epidermal growth factor (EGF) and its receptor (EGFR) in oocytes and cumulus cells from goat COCs recovered by laparoscopy after standard [multi-dose follicle-stimulating hormone (FSH)] or one-shot (single dose FSH plus eCG) treatments. No differences were observed among the number of recovered and morphologically graded COCs or the IVM rates for both gonadotropic treatments. However, the standard protocol produced COCs with higher EGFR expression in the cumulus cells than the one-shot treatment. Additionally, EGF mRNA levels were less than EGFR mRNA levels, and they did not differ among COCs from both treatments. However, during maturation, the EGF transcripts increased in oocytes derived only from the standard protocol. Interestingly, IVM strikingly increased EGFR expression in oocytes and cumulus cells but not in oocytes that fail in first polar body extrusion, irrespective of hormonal treatment. These results appear to be related to the resumption of meiosis and suggest that EGF may act through the cumulus cells or directly on the oocyte receptor.

Pronuclear embryo yield in Canindé and Saanen goats for DNA microinjection.

The objective of this study was to examine the effect of donor breed on pronuclear-stage embryo yield to be used for DNA microinjection in a transgenesis goat program. Twelve Canindé and twelve Saanen goats were heat synchronized using a progestagen-cloprostenol treatment. Forty-eight hours before the sponge removal, superovulation was induced with a total administration of 4.4 mg/kg bodyweight NIH-FSH-P1, given twice daily in decreasing doses over 3 days. In addition, goats received 100 µg of GnRH and they were hand-mated at 36 and 48 h after progestagen removal. Embryo recovery was performed by oviduct flushing at 72 h after sponge removal. Embryos were microinjected with a DNA construct and noticeable swelling of the nuclei was the criterion for successful microinjection. The total diameter, cytoplasm diameter, zona pellucida thickness and pronuclei diameter were measured for each microinjected embryo. A higher (p < 0.05) percentage of fertilized ova was observed in Canindé (89.9%) than Saanen (36.2%) goats. In addition, Canindé donors produced a higher percentage of pronuclear embryos when compared with Saanen: 72.5% vs 20.6% (p < 0.05), respectively. Successful microinjection was verified in 96.7% and 73.3% of times in Canindé and Saanen embryos, respectively (p < 0.05). Significant differences were observed for all morphometric parameters except for cytoplasm diameter. In conclusion, under our study experimental conditions, Canindé were more efficient than Saanen goats concerning the pronuclear embryo yield and manipulation. The use of Canindé goats in transgenesis programs could be increase the interest in their breeding and could be contribute to saving them from extinction.

Ion-exchange chromatography used to isolate a spermadhesin-related protein from domestic goat (Capra hircus) seminal plasma

Mammalian seminal plasma contains among others, proteins called spermadhesins, which are the major proteins of boar and stallion seminal plasma. These proteins appear to be involved in capacitation and sperm-egg interaction. Previously, we reported the presence of a protein related to spermadhesins in goat seminal plasma. In the present study, we have further characterized this protein, and we propose ion-exchange chromatography to isolate this seminal protein. Semen was obtained from four adult Saanen bucks. Seminal plasma was pooled, dialyzed against distilled water and freeze-dried. Lyophilized proteins were loaded onto an ion-exchange chromatography column. Dialyzed-lyophilized proteins from the main peak of DEAE-Sephacel were applied to a C2/C18 column coupled to an RP-HPLC system, and the eluted proteins were lyophilized for electrophoresis. The N-terminal was sequenced and amino acid sequence similarity was determined using CLUSTAL W. Additionally, proteins from DEAE-Sephacel chromatography step were dialyzed and submitted to a heparin-Sepharose high-performance liquid chromatography. Goat seminal plasma after ion-exchange chromatography yielded 6.47 +/- 0.63 mg (mean +/- SEM) of the major retained fraction. The protein was designated BSFP (buck seminal fluid protein). BSFP exhibited N-terminal sequence homology to boar, stallion and bull spermadhesins. BSFP showed no heparin-binding capabilities. These results together with our previous data indicate that goat seminal plasma contains a protein that is structurally related to proteins of the spermadhesin family. Finally, this protein can be efficiently isolated by ion-exchange and reverse-phase chromatography.

Effect of age of donor on embryo production in Morada Nova (white variety) ewes participating in a conservation programme in Brazil.

In order to evaluate embryo production in Morada Nova (white variety) ewes superovulated with porcine follicle-stimulating hormone, 20 cycling ewes were used as embryo donors and allocated into two groups according to age: group 1 (ewes aged 1-2 years; n = 9) or group 2 (ewes aged 3-4 years; n = 11). Embryo recovery was performed by laparotomy 5-6 days after oestrus. The evaluation of embryos was made under stereomicroscope according to International Embryo Transfer Society rules. The overall recovery rate was 64.6% (5.0 +/- 0.7 structures per ewe) and 86.3% of the recovered structures were fertilized. Group 1 was superior (p < 0.05) to group 2 according to recovered (6.6 +/- 0.9 vs 3.6 +/- 0.8) and fertilized structures (5.6 +/- 1.1 vs. 3.5 +/- 0.7) per ewe. In conclusion, the ovarian response and the embryo production in Morada Nova (white variety) sheep subjected to a standard superovulation treatment were considered satisfactory. In addition, the use of multiple ovulation and embryo transfer in younger ewes ( < or = 2 years old) of this sheep breed appears to be an efficient tool to accelerate the preservation of the Morada Nova (white variety) breed, since younger ewes are as responsive as older ones.

Responsiveness to progestagen-eCG-cloprostenol treatment in goat food restricted for long period and refed.

For 6 months, 10 adult Saanen crossbred goats were fed undernutrition diet (70% maintenance), and finally five goats were refed for 6 weeks with 150% maintenance. In all animals oestrus was synchronized using 45 mg FGA vaginal sponge for 11 days, 300 IU eCG and 50 microg cloprostenol 48 h prior to sponge removal. From oestrus onset, during a 24-h period, blood samples were collected for oestradiol and NEFA assay. Ovulation was verified by laparoscopy 3 days after sponge removal. Body mass loss was 18.62 +/- 3.03% of initial weight and in refed goats body weight recovery was 90.63 +/- 3.56%. NEFA level was higher in restricted goats (p < 0.05). Fifty per cent of underfed goats (2/4) and all refed goats (4/4) exhibited oestrus and ovulation. Significant relationship (p < 0.05) was found between weight loss and the interval sponge removal-oestrus onset (r = 0.91) or ovulation rate (r = 0.70). Only in the refed group was the ovulation rate related to the oestradiol amount (r = 0.99) (p < 0.05). Collectively results showed that a short period of improved feeding re-established the responsiveness of oestrus synchronization in chronically fasted goats.

Hormonal treatments for the synchronisation of oestrus in dairy goats raised in the tropics.

In tropical areas, local goats are often reported as being able to reproduce throughout the year, whereas an influence of season is found to be a factor when importing different dairy breeds. In these areas, oestrus synchronisation in goats is of interest for both technical (synchronisation of kidding, adjustment to forage availability or to continuous milk supply) and genetic reasons (dissemination of improved genotypes by AI). The use of a progestagen vaginal sponge combined with equine chorionic gonadotrophin (eCG)-cloprostenol injections remains an efficient tool to achieve synchronisation in temperate and tropical zones. However, the oestrus synchronisation treatments currently used for goats in tropical regions were originally developed for goats bred in temperate regions. For this reason, several alternative possibilities for improving the efficiency of the hormonal treatment are evaluated. Oestrus synchronisation with luteolytic agents is efficient (resulting in more than 70% of goats in oestrus) and it takes into account female cyclicity. In developing regions of the tropics, the use of buck teasing appears to be a promising approach to control oestrus and ovulation. The use of this technique provides 60% of females in oestrus within 5 days of introducing the bucks. Considering the availability of nutrients as the ultimate regulator of reproduction in the tropics, the control of nutritional condition is essential before the use of hormonal treatments for oestrus synchronisation in goats bred in these regions takes place.

Pseudopregnancy in Saanen goats (Capra hircus) raised in Northeast Brazil.

The prevalence of pseudopregnancy over 44 months was investigated in 23 Saanen goats raised in Northeast Brazil during continuous oestrous cycling (cyclic group) or after synchronization of oestrus (synchronized group). The goats were monitored by ultrasonography and their plasma progesterone profile. The overall prevalence of pseudopregnancy was 30.4% (7/23). In the cyclic group, 28.6% (4/14) of goats showed pseudopregnancy, while in the synchronized group the prevalence was 33.3% (3/9). There was no significant difference between the groups (p>0.05). The mean (+/- SD) length of pseudopregnancy, as shown by the progesterone profile, was 121.6 +/- 33.5 days, ranging from 70 to 155 days. The study defined the prevalence of pseudopregnancy in Saanen goats raised in Northeast Brazil for the first time. This finding identified a major problem for this breed, as without treatment such animals remain unproductive until the spontaneous resolution of the condition. More research seems desirable to ascertain the prevalence of this condition in other breeds in this region of Brazil.

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer

Birth of normal kids after microinjection of pronuclear embryos in a transgenic goat (Capra hircus) production program in Brazil.

This pilot project was designed to determine if normal kids could be produced after microinjection in pronuclear embryos and subsequent transfer to recipients in a transgenic goat program in Brazil. Twelve donors of the Saanen breed and 17 recipients of an undefined breed were used. The estrus of both donors and recipients was synchronized by a standard progestagen treatment and superovulation obtained by six pFSH injections. Donors in estrus were mated with fertile Saanen bucks. Zygotes were recovered surgically by flushing oviducts. The recovered zygotes with visible pronuclei were microinjected with 500 to 1000 copies of the human G-CSF gene. Two or four embryos were surgically transferred into the oviducts of recipients. One recipient became pregnant and two kids were born. No transgenic goat was identified after PCR analysis. Even though transgenic goats were not obtained, this experiment establishes the basis of a synchronization and superovulation regimen for use in goats raised in Brazil, for the purpose of collecting and manipulating the pronuclear embryos. This project also showed that microinjected one-cell goat embryos can survive to produce live young following surgical transfer.

Isolation and partial characterisation of a protein from buck seminal plasma (Capra hircus), homologous to spermadhesins.

Spermadhesins are a family of secretory proteins expressed in the male genital tract of pig, horse and bull. Their function and structure have been widely studied, especially those isolated from boar. However, there are no data concerning spermadhesins isolated from buck. Buck seminal plasma was collected and subjected to ion exchange chromatography on DEAE-Sephacel column followed by chromatography in a C18 column coupled to a HPLC system. The purification of the protein was determined by SDS-PAGE and MALDI-TOF analysis exhibiting a molecular mass of 12.5 KDa and showed to be structurally homologous to spermadhesins from boar and stallion.