Júlio César Ferraz Jacob

Combination of hCG and Deslorelin Acetate on the Induction of Ovulation in Mares: Changes in Follicular Fluid Protein Profile

The composition of follicular fluid (FF) is essential for the proliferation and differentiation of granulosa cells in addition to processes related to rupture of the follicular wall, maturation and fertilization of the oocyte and luteinization. In the mare, there are few studies evaluating FF proteome (FAHIMINIYA, Prot Sci, 9:1, 2011; PETRUCCI, J Eq Vet Sci, 34:115, 2014). The ability to induce ovulation in a reliable way is important in equine reproductive management in different situations the main ovulation-inducing agents used are hCG and deslorelin. The aim of this study was to compare the protein profile of FF on induced ovulation of mares with hCG or with the combination of hCG and deslorelin acetate. Fourteen mares were used (3-12 years). Following the observation of follicles ≥ 35 mm and with endometrial edema, the mare was submitted to the induction protocols: Group H 1000 UI, IV, of hCG or Group HG 1000 UI hCG, IV, + 1,5 mg of Deslorelin acetate, IM. In the subsequent cycle mares were submitted to a protocol different from the previous cycle. Samples were collected by transvaginal aspiration 32 h after induction and submitted to the quantification of proteins by the Bradford method. Two-dimensional electrophoresis was performed in 12.5% polyacrylamide gel and stained with Coomassie G250, scanned and analyzed using PDQuest v.8.0.1. Spots with significant differences in relative abundance between group H and HG were cut out submitted to trypsin digestion and mass spectrometry. In this study the total protein concentration in the mare FF from Group HG were higher (73.07 ± 6.42 mg/ml) than those induced with hCG alone (63.97 ± 6.97 mg/ml). Comparative analysis showed a significant difference in the abundance of five spots between groups. Two Alpha-1-antiproteinase 2 (A1AT2), the Serotransferrin (TF) and Antithrombin III (ATIII) had lower relative expression in group HG and the Haptoglobin (HP) showed greater abundance in the same group. The lowest expression of A1AT2 at the final moment of follicular maturation prior to ovulation is likely related to the need for lower inhibitory action on the proteolytic activity allowing fine adjustment that controls the ECM degradation, inflammation and the coagulation cascade (BIANCH, J Prot, 90:61, 2013). ATIII is also serine-type endopeptidase inhibitor activity. The last protein less expressed in the group HG was TF. Increase in cellular iron levels stimulates the expression of some MMPs that degrade the ECM. There are reports of increased transferrin in granulosa cells and oocyte follicles in more advanced stages of maturation, which could explain the reduction in FF transferrin. Haptoglobulin showed increased abundance in the group HG and exerts anti-inflammatory action due to inhibition of oxidative damage. These proteins are probably related to the final events of oocyte/follicle maturation that trigger ovulation and subsequent luteinization.

Can sperm selection, inseminating dose and artificial insemination technique influence endometrial inflammatory response in mares?

Abstract The aim of this study was to investigate the influence of sperm concentration, sperm quality, and semen deposition site on uterine inflammatory response in mares. Twenty‐five mares with healthy reproductive tracts were used for artificial inseminations (AIs) using frozen‐thawed semen and divided into 5 experimental groups: Control—insemination with 800 × 106 total spermatozoa in the body of the uterus (n = 5); G2—insemination with 40 × 106 total motile sperm (TMS) into uterine body (n = 5); G3—insemination with 40 × 106 TMS at the uterotubal junction by hysteroscopy (HI) (n = 5); G4—insemination with 40 × 106 TMS at the uterotubal junction by HI, with frozen‐thawed semen selected by Percoll gradient (n = 5); G5—insemination with 40 × 106 total motility sperm into uterine body, using frozen‐thawed semen selected by Percoll gradient (n = 5). Number of polymorphonuclear neutrophils in uterine cytology was observed at 8 and 24 hours after AI. A reduced inflammatory endometrial reaction (P < .05) was observed, both at 8 and 24 hours, in mares inseminated with low sperm dose (40 × 106) compared with those inseminated with regular semen dose (800 × 106), with the exception of G4 that did not differ from the control group. Sperm selection by Percoll gradient did not influence the intensity of endometrial inflammatory response (P > .05) regardless of the type of insemination (conventional or hysteroscopic). In addition, mares inseminated by hysteroscopy showed a greater local inflammatory reaction (P < .05) in relation to those inseminated into uterine body. In conclusion, reduction of sperm insemination dose may be an alternative method to decrease endometrial inflammatory response in mares. However, hysteroscopic insemination and the current protocols used for sperm selection by Percoll did not show efficient results to reduce endometrial inflammation in mares. Further studies about sperm selection and HI insemination techniques are necessary to determine the benefits and the appropriate protocol to be performed in barren mares. HighlightsEndometrial uterine inflammation in mares depends on the number of inseminated sperm.Sperm selection by Percoll before insemination does not reduce the endometrial inflammation in mares.Hysteroscopic insemination does not reduce the endometrial inflammation in mares.

Use of different pressures for transvaginal follicular aspiration in mares

The success of transvaginal follicular aspiration in mares can be influenced by several factors, such as vacuum pump pressure levels. The present study aimed to investigate the effect of different negative pressures (150, 280 and 400mmHg) of the vacuum pump on the oocyte recovery in the mares. The mares (n=10) were undergoing follicular aspiration using three different negative pressures for three consecutive estrous cycles as follows: G150 = 150mmHg (n = 10); G280 = 280mmHg (n = 10); G400 = 400mmHg (n = 10). Every estrous cycle, the group that the mare would participate was drawn, and each animal participated once in each group. Only preovulatory follicle was used, about 30 to 36 hours after application of hCG. To compare the results, the chi-square test was used (5% significance) and Fisher exact test, when recommended. Thirty preovulatory follicles (diameter 36.1±1.80mm) were aspirated and ten oocytes were recovered (33.3%). There was no statistical difference between the experimental groups (p=0.59). Thus, accord to the results observed in this study, we could conclude that the negative pressure of the vacuum pump used was not efficient to increase oocyte recovery.ABSTRACT The success of transvaginal follicular aspiration in mares can be influenced by several factors, such as vacuum pump pressure levels. The present study aimed to investigate the effect of different negative pressures (150, 280 and 400mmHg) of the vacuum pump on the oocyte recovery in the mares. The mares (n=10) were undergoing follicular aspiration using three different negative pressures for three consecutive estrous cycles as follows: G150 = 150mmHg (n = 10); G280 = 280mmHg (n = 10); G400 = 400mmHg (n = 10). Every estrous cycle, the group that the mare would participate was drawn, and each animal participated once in each group. Only preovulatory follicle was used, about 30 to 36 hours after application of hCG. To compare the results, the chi-square test was used (5% significance) and Fisher exact test, when recommended. Thirty preovulatory follicles (diameter 36.1±1.80mm) were aspirated and ten oocytes were recovered (33.3%). There was no statistical difference between the experimental groups (p=0.59). Thus, accord to the results observed in this study, we could conclude that the negative pressure of the vacuum pump used was not efficient to increase oocyte recovery. Keywords: oocyte, subfertility, preovulatory follicle, hCG

Reproductive efficiency of asymptomatic Theileria equi carriers mares submitted to an embryo transfer program

This study aimed to assess and evaluate the effects of Theileria equi infection on embryonic recovery, gestation and early embryonic loss. Thirteen Mangalarga Marchador Theileria equi positive donors (diagnosed through nested-PCR) and 40 embryos receptors were used. Donors were submitted to two embryo collections in two consecutive estrous cycles (GId); after, the same mares were treated with imidocarb dipropionate (1.2mg/kg IM.) in order to collect more embryos in two more estrous cycles (GIId). Receptors were divided into two groups (control and with treated) with 20 animals each, where one group was the control (GIr) and the other one (GIIr) treated with 1.2mg/kg IM of imidocarb dipropionate assessing the gestation rate at 15, 30, 45 and 60 days. After 52 embryo collections, the embryonic recovery rates were 53.84% (14/26) and 65.38% (17/26) (p> 0.05) for GId and GIId, respectively. The gestation rate was 70% (14/20) (p>0.05) at 15, 30, 45 and 60 days in group GIr and for GIIr was 85% (17/20) (p>0.05) at 15 days, 80% (16/20) (p>0.05) at 30, 45 and 60 days. The treatment with imidocarb dipropionate did not cause significant improvement in the reproductive efficiency at an ET program.