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In order to enhance the use of biotechnology in females it is necessary to have good knowledge of the ovarian events, and thus enable the manipulation of the estrus cycle to concentrate ovulations in an established period of time. In this experiment, we evaluated the ovary response and corpus luteum (CL) parameters in a short-duration protocol associated or not to lecirelin (L)(GnRH) and/or estradiol benzoate (EB). Twenty-four cyclic Santa InÊs ewes were divided into 4 groups: Control animals (G1; n = 6) received 45 μg of d-cloprostenol (Prolise®, Tecnopec, Sao Paulo, Brazil) on Day 0. Intravaginal sponges containing medroxyprogesterone acetate (MAP60®, Tecnopec) were inserted on Day 3. Then on Day 7 sponges were removed and 400 IU of eCG and 45 μg of d-cloprostenol were administered. G2 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1; G3 (n = 6) animals underwent the same protocol as G1 but with administration of 25 μg of L (Gestran plus®, Tecnopec) 30 h after sponge withdrawal; G4 (n = 6) animals underwent the same protocol as G1 but with administration of 1 mg of EB on Day 1 and 25 μg of L 30 h after sponge withdrawal. Ewe ovaries were assessed via transrectal ultrasound (US), and CL were measured 4 days after ovulation. On the same day blood samples were taken to measure plasma progesterone levels via radioimmunoassay. Twenty-three out of 24 animals ovulated. One ewe from G4 that did not ovulate was taken out of the experiment. The average size of the largest pre-ovulatory follicle was 7.3 mm (G1), 7.0 mm (G2), 6.8 mm (G3), and 7.4 mm (G4) (P > 0.05). There were 1.8, 1.2, 2.0, and 1.4 ovulated follicles per animal in G1, G2, G3, and G4, respectively. The results for estrus start and duration after sponge withdrawal were G1: 32/38.6; G2: 37/69.3; G3: 29.3/37.3; G4: 44/68 h, respectively. Ovulation time span was G1: 65.3; G2: 88; G3: 53.3; G4: 82.4 h after sponge withdrawal. There was a high correlation of ovary mensuration and progesterone level (r = 0.64; P < 0.0001) on the fourth day after ovulation. Progesterone levels after ovulation were 2.37 ng mL-1 (G1); 1.5 ng mL-1 (G2); 3.22 ng mL-1 (G3) and 1.99 ng mL-1 (G4), being higher in G3 than in G2 (P < 0.05). It was seen that the EB is prejudicial to the CL function. Despite the fact that there was no significant difference of progesterone levels within G1, G3, and G4, animals in G3 displayed higher levels of progesterone; hence, there is a need for further studies using a larger number of animals and fertility test. CNPq e Tecnopec.

Eleven semen samples from Santa Ines breed rams were cryopreserved with the objective of studying the effect of the dimethyl acetamide (DMA, 3%) or glycerol (GL, 6%) in an extender (Tris) with or without trehalose (TRE, 100 mOsmol) on post-thaw sperm viability and fertility. After the evaluation, the semen was diluted in extenders (TRIS+GL6%; TRIS+GL6%+TRE; TRIS+DMA3%; Tris+DMA3%+TRE) and then frozen. The post-thaw kinetic sperm parameters evaluated were the percentage of total motility (TM) and rapid spermatozoa (RAP), track speed (VCL), lateral amplitude of head (ALH), and linearity (LIN). The sperm viability was verified using the fluorescence markers propidium iodide (PMI, plasmatic membrane integrity), JC-1 (MMP, mitochondrial membrane potential), and FITC-PSA (ACI, acrosomal integrity). The spectrum of DNA sperm damage was assessed by single cell gel electrophoresis (Comet assay), and the measurements performed were tail moment (TMOM) and tail intensity (TINT; Comet Assay II software, Perceptive Instruments, Haverhill, UK). The sheep estrus synchronization protocol (n = 170) was 12 days of sponge with 60 mg of medroxyprogesterone acetate and 300 UI of eCG i.m. at sponge withdrawal. The ewes were inseminated on 57.5 h after sponge removal with 150 × 106 of live spermatozoa. The conception rates (CR) were verified for ultrasonography 45 days after insemination. The statistical analysis was performed using SAS software (SAS Institute Inc., Cary, NC, USA). The means (±SEM) of in vitro post-thaw sperm parameters and fertility rates are shown in Table 1. The greatest (P < 0.05) rates of TM and RAP for the GLY groups were observed in relation to the DMA ones. It was also verified the inferiority (P < 0.05) in the VCL and ALH for the trehalose extenders in both groups with GLY and DMA. The values (%) of TINT and TMOM were 14.9 and 03.9, 15.8 and 03.8, 14.7 and 02.9, and 16.6 and 04.4, respectively, for TRIS+GL6%, TRIS+GL6%+TRE, TRIS+DMA3%, and TRIS+DMA3%+TRE (P > 0.05). The integrity rates of all sperm membranes were superior for TRIS+GL6% in relation to TRIS+DMA3%. In conclusion, the DMA had apoor cryoprotective effect for ram semen. The addition of trehalose promoted negative effect on sperm kinetics, although it improved levels of PMM post-thawing in the DMA extender. The conception rate did not present a difference (P > 0.05) among the groups. Table 1.