Resumo em Inglês:

Abstract Artificial insemination (AI) was the first important biotechnology applied to improve the genetics of farm animals. It allows the rapid and massive diffusion of desirable characteristics of males with high productive potential. We describe the different types of estrus induction and synchronization techniques and the use of the AI with fresh, chilled or frozen semen. Through the adequacy of the protocols of estrus synchronization and AI to the different production systems, the efficient use of reproductive techniques is possible, reaching acceptable pregnancy rates. Summary of reproductive results obtained using cervical and laparoscopic AI are presented.

Resumo em Inglês:

Abstract Metronidazole is an antiprotozoal and antibacterial used in gynecology and obstetrics for the treatment of parasitic infections. However, despite having clinical use for more than three decades, questions about the safety of its use during pregnancy is not well understood. Thus, the present study evaluated the effect of metronidazole on placental and fetal development in pregnant rats. Metronidazole was orally administered by gavage at a dosage of 130 mg/kg for 7 and 14 days. Morphological analysis, morphometry and immunohistochemistry were performed at the implantation sites and placentas with 14 days of development. The results showed that in the treated group there was a significant reduction in the number of implantation sites, total placental disc area and constituent elements of the labyrinth and spongiotrophoblast layers. Histochemical analysis revealed no significant changes in the content of collagen, elastic and reticular fibers. The TUNEL test showed apoptotic activity in the implantation sites and placentas with 14 days of development independent of the treatment. There was no evidence of malformation in the neonates. However, there was a significant reduction in the number and weight of neonates in the group treated with metronidazole when compared to the control group. Thus, it is concluded that the administration of 130 mg/kg of metronidazole during pregnancy in rats, in addition to interfering with the number of implanted embryos, promotes changes in placental structure and interferes with fetal development. This suggests that this drug should be used with caution during pregnancy.

Resumo em Inglês:

Abstract The objective of this study was to test the efficiency of powdered coconut water (ACP-406®) base-medium without or with the addition of supplements on in vitro culture of isolated goat secondary follicles. Follicles were cultured for 18 days in α-MEM or in ACP-406®, both without supplements (referred to as α-MEM and ACP, respectively), or both supplemented with BSA, insulin, transferrin, selenium, glutamine, hypoxanthine, and ascorbic acid (referred to as α-MEM+ and ACP+). Follicular morphology, antrum formation, follicular and oocyte diameter, levels of glutathione (GSH), and chromatin configuration after in vitro maturation were evaluated. At the end of culture, ACP-406® base-medium (without or with supplements) showed a higher (P < 0.05) percentage of normal follicles than α-MEM (without or with supplements). Antrum formation was similar among α-MEM+, ACP and ACP+, and significantly higher than α-MEM without supplements. The follicular diameter was greater in ACP+ than α-MEM, and similar to other treatments. Moreover, fully and daily grown rates were higher (P < 0.05) in ACP-406® base-medium (without or with supplements) than α-MEM (without or with supplements). Levels of GSH were similar between ACP+ and α-MEM+ treatments. Both ACP+ and α-MEM+ allowed meiotic resumption without a significant difference between the two groups. In conclusion, supplemented ACP-406® base-medium maintained follicular survival and promoted the development as well as meiotic resumption of isolated goat secondary follicles cultured in vitro for 18 days.

Resumo em Inglês:

Abstract The transforming growth factors beta (TGFβ) are local factors produced by ovarian cells which, after binding to their receptors, regulate follicular deviation and ovulation. However, their regulation and function during corpus luteum (CL) regression has been poorly investigated. The present study evaluated the mRNA regulation of some TGFβ family ligands and their receptors in the bovine CL during induced luteolysis in vivo. On day 10 of the estrous cycle, cows received an injection of prostaglandin F2α (PGF) and luteal samples were obtained from separate groups of cows (n= 4-5 cows per time-point) at 0, 2, 12, 24 or 48 h after treatment. Since TGF beta family comprises more than 30 ligands, we focused in some candidates genes such as activin receptors (ACVR-1A, -1B, -2A, -2B) AMH, AMHR2, BMPs (BMP-1, -2, -3, -4, -6 and -7), BMP receptors (BMPR-1A, -1B and -2), inhibin subunits (INH-A, -BA, -BB) and betaglycan (TGFBR3). The mRNA levels of BMP4, BMP6 and INHBA were higher at 2 h after PGF administration (P<0.05) in comparison to 0 h. The relative mRNA abundance of BMP1, BMP2, BMP3, BMP4, BMP6, ACVR1B, INHBA and INHBB was upregulated up to 12 h post PGF (P<0.05). On the other hand, TGFBR3 mRNA that codes for a reservoir of ligands that bind to TGF-beta receptors, was lower at 48 h. In conclusion, findings from this study demonstrated that genes encoding several TGFβ family members are expressed in a time-specific manner after PGF administration.

Resumo em Inglês:

Abstract This study evaluated a powdered coconut water solution (ACP 406®) as a base culture medium on the in vitro survival and development of in situ goat preantral follicles. The ovarian fragments were either immediately fixed in Carnoy solution (non-cultured control) or individually cultured for 2 or 6 days. The following culture media (all containing 100 μg/mL penicillin and 100 μg/mL streptomycin) were evaluated: α-MEM (α-MEM alone, without additional supplementation); α-MEM+ (supplemented α-MEM); ACP (ACP®406 alone); or ACP+ (supplemented ACP®406). Additional supplementation includes: 1.25 mg/mL bovine serum albumin, 10 μg/mL insulin, 5.5 μg/mL transferrin, 5 ng/mL selenium, 2 mM glutamine, and 2 mM hypoxanthine. The endpoints (i) follicular morphology; (ii) development; (iii) estradiol production; and (iv) reactive oxygen species (ROS) were recorded. Data were analyzed using chi-square, Turkey, t-test or One-Way ANOVA. Differences were considered significant when P < 0.05. At day 2 of culture, a greater (P < 0.05) percentage of morphologically normal follicles was observed between ACP+ and ACP treatments. Moreover, at day 2 of culture, no hormonal difference (P < 0.05) was observed between ACP+ and both α-MEM treatments. At day 6 of culture when ACP and α-MEM treatments were compared the percentage of healthy follicles were similar (P > 0.05) among treatments. Overall, all treatments had lower primordial follicles (P < 0.05) accompany by greater developing follicles (P < 0.05) percentages than non-cultured control treatment, indicating primordial follicle activation. However, at day 6 of culture, the percentage of primordial follicle development were similar (P > 0.05) among the treatments. Likewise, no differences (P > 0.05) were observed for ROS production and follicular and oocyte diameters among treatments. Therefore, ACP+ has the equivalent efficiency to MEM+ in maintaining the survival and development of goat preantral follicles, representing an alternative plant-based low-cost culture medium for in vitro culture.

Resumo em Inglês:

Abstract In this study, 252,798 lactations on 108,077 cows in 433 herds were used to determine the association between gestation length (GL) and lactation performance, lactation curve, calf birth weight and dystocia in Holstein dairy cows in Iran. The GL averaged 278.1 ± 5.41 d, was categorized as short (SGL; at 1 SD below the population mean), average (AGL; the population mean ± 1 SD), or long (LGL; at least 1 SD above the population mean). Factors including parity, calf gender and calving season were associated with the GL. Primiparous cows with SGL had less lactation performance than those with longer GL; however, there was no difference between those with AGL and LGL. Multiparous cows with longer GL always had more partial and 305-d lactation performance. Primiparous cows with SGL produced less milk at the beginning of lactation and at the peak than those with AGL or LGL; inverse trends were found for lactation persistency, upward and downward slopes of the lactation curve. Within multiparous, a direct relationship was found between GL and the peak yield, where cows with longer GL always produced more milk at the peak. Multiparous cows with SGL produced less milk at the beginning of lactation, reached their peaks later, had higher lactation persistency and showed a lower upward slope of lactation curve than those with AGL or LGL. There was a direct relationship between GL and calf birth weight, where cows with longer GL had calves with more weight at the birth. Within primiparous, cows with SGL had the lowest and those with LGL had the highest rate of dystocia. However, multiparous cows with AGL had a lower rate of dystocia than those with SGL or LGL. Although there was a direct relationship between GL and lactation performance, intermediate GL seems optimal when considering dystocia.

Resumo em Inglês:

Abstract The main goal of this preliminary study was to determine and compare ultrasonographic characteristics of the mammary gland in two genotypes of ewes varying in milk productivity at 2, 3 and 4 weeks after lambing. Ultrasonographic images of the udder were obtained using the 5.0- and 7.5-MHz transducers, in axial and coronal planes, in four low milk-yielding Polish Mountain sheep and six high milk-yielding Olkuska ewes. All ultrasonograms were subjected to computerized image analyses using commercially available image analytical software (Image ProPlus®; Media Cybernetics Inc., San Diego, CA, USA) to determine numerical pixel values (NPVs) and heterogeneity (pixel standard deviation-PSD) of the mammary gland parenchyma. During the 28-day period post-partum, the Olkuska sheep exceeded (P < 0.05) Polish Mountain ewes in milk productivity (31.6 ± 2.7 l and 25.0 ± 4.2 l, respectively; means ± SEMs) as estimated by the mean weight gains of suckling lambs. In animals examined with the 5.0-MHz transducer, mean NPVs of the mammary gland parenchyma in Olkuska ewes and mean PSD in both genotypes of ewes were lower (P < 0.05) before than after milking. In addition, PSD recorded both before and after milking were lower (P < 0.05) in the Polish Mountain compared with Olkuska breed. Mean PSD values for the mammary gland were less (P < 0.05) before than after milking in Polish Mountain ewes and they were greater (P < 0.05) in Olkuska compared with Polish Mountain ewes examined with the 7.5-MHz probe after milking. It can be concluded that milk quantity, histomorphology of the udder and ultrasound transducer frequency may all impinge on the echotextural characteristics of the mammary parenchyma in different breeds of sheep. Our observations warrant future studies of correlations between milk composition, mammary gland histophysiology and ultrasonographic image attributes of the mammary gland in ruminants.

Resumo em Inglês:

Abstract This study aimed to assess the influence of live body weight (LBW) and age on reproductive performance in buffalo heifers synchronized by different treatments. The study was carried out on 146 Mediterranean buffalo heifers (mean age 25.3±13.4 months, LBW 424±47 kg), divided into 2 homogeneous groups and synchronized by Ovsynch-TAI Program (OVS; n = 72) or double prostaglandin administered 12 days apart (PGF; n = 74). All the buffaloes were inseminated twice and follicle dimensions and ovulation rate (OR) were assessed by ultrasound 24 and 48 h post-insemination. Pregnancy was assessed on day 25, 45 and 90 post-insemination and the incidence of late embryonic (LEM) and fetal (FM) mortality were respectively recorded. Data were analyzed by ANOVA, Chi-square test and multiple logistic regression. The LBW was significantly (P<0.05) higher in inseminated animals, compared to those that did not respond to the treatments (450.0±3.2 vs. 423.2±9.6 kg in inseminated and not inseminated heifers, respectively). Total OR was similar between groups, although OR at 24 h tended to be higher (P = 0.06) in OVS (86.7 vs. 72.9% in OVS and PGF, respectively). A (P<0.01) higher LBW was observed in ovulated heifers of PGF, while no differences were recorded in OVS. LBW affected OR (odds ratio = 1,032; P<0.05) only in PGF, while no effects were recorded in OVS. Total pregnancy rate, LEM and FM were similar between groups. In conclusion, the LBW would be considered before including buffalo heifers in a synchronization program and both synchronization treatments can be useful.

Resumo em Inglês:

Abstract The aim of this study was to assess the usefulness of nanowater (NW; water declusterized using cold plasma treatment) as a diluent for a commercial boar semen extender during the 15-day storage (Days 1 to 15) at 16-18 °C. Ejaculates collected from 8 boars were subjected to the standard evaluation and then diluted in the extender prepared with deionized water (DW) or NW to a final concentration of 3×109 spermatozoa/ml. The proportion of defective spermatozoa increased (P<0.05) from Day 10 to Day 15 of storage (22.8±16.6% to 41.8±26.4% in DW group and 18.6±11.7% to 34.8±25.4% in NW group) and it was significantly greater in DW group compared with NW group on Days 5 and 10 due mainly to a greater (P<0.05) number of mid-piece defects in semen stored in the DW-containing extender. Sperm progressive motility decreased (P<0.05) in both groups between Days 2 and 6, Days 6 and 10, and Days 10 and 12, whereas the percentage of motile spermatozoa declined (P<0.05) to Day 14 only in NW group. Sperm motility was greater (P<0.05) in NW group compared with DW group from Day 5 to Day 13. A decline in sperm progressive motility below 40% in all semen samples occurred by Day 11 in DW group and by Day 12 in NW group. The mean survival time of sperm at 37 °C ex situ was greater in NW group than in DW group on Day 5 (314±87 min compared with 284±87 min) and Day 10 (223±34 min compared with 182±27 min; NW group compared with DW group, respectively). There were no differences (P>0.05) between the two groups in the concentrations of alkaline phosphatase and aspartate aminotransferase in semen extender. To summarize, the use of NW as an extender diluent exerts cytoprotective effects on boar spermatozoa and delays a decline in sperm progressive motility.

Resumo em Inglês:

Abstract This study aimed to investigate the effects of bacterial endotoxin lipopolysaccharide (LPS) on hormone production and gene expression in duck Leydig cells and its underlying mechanisms. Leydig cells were collected from 200-day-old mallard ducks and divided into five treatment groups (0, 50, 100, 200, and 400 ng/mL LPS). After treatment with LPS for 6, 12, 24, and 48 h, testosterone, activin, and inhibin levels in the cell supernatants were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression levels of testosterone synthesis-related genes, including steroidogenic acute regulatory protein (StAR), 3-beta-hydroxysteroid dehydrogenase (3β-HSD), and cytochrome P450 aromatase (P450arom), and reproductive-related genes, including gonadotropin-inhibitory hormone receptor (GnIHR), follicle stimulating hormone receptor (FSHR), and luteinizing hormone receptor (LHR) were detected using quantitative real-time polymerase chain reaction (qRT-PCR). We successfully isolated and cultured duck Leydig cells with cell purity above 90%. Compared with the control group, the levels of testosterone, activin, and inhibin secreted in Leydig cells decreased gradually with increasing LPS concentration. After treatment with LPS, the expression of StAR and 3β-HSD genes in Leydig cells was upregulated at 12 h, and that of GnIHR was upregulated at 24 h; whereas the expression of FSHR and LHR was reduced at 24 h. This study indicates that LPS can inhibit the secretion of hormones and regulate the expression of related genes in duck Leydig cells.

Resumo em Inglês:

Abstract The aim of this study was to evaluate the impact of successive bovine testicular punctures using different needle sizes. Fifteen bulls were submitted to testicular needle aspiration (TNA) in the left and right testis using 18-gauge (40×12mm) or 22-gauge (25×7mm) needles, respectively, once every 30 days. Animals were randomly divided into three groups, which were submitted to bilateral orchiectomy two days after the last puncture. Group 1 (G1): only one puncture (n=5); Group 2 (G2): three consecutive punctures in a period of three months (n=5); Group 3 (G3): six consecutive punctures in a period of 6 months (n=5). Fragments from the medial portion of the testicular parenchyma were excised and fixed in Bouin’s fluid for histological analysis. No differences were observed in the percentage of seminiferous tubules degeneration between G1, G2 and G3 (P>0.05). Higher amounts of erythrocyte were found in G1 and G2 groups compared to G3, in the intra- and intertubular tissue (P<0.05). There was no interaction between the needle gauge and the occurrence of testicular damage in animals submitted to one (G1) or three (G2) punctures. However, a higher percentage of tubular degeneration was associated to 18-gauge compared to 22-gauge fine needles in G3. In conclusion, multiple testicular needle aspiration can be safely conducted using fine needles. Large needles are recommended only for a single TNA, since multiple punctures may result in increased tubular degeneration and compromise testicular architecture and functionality.

Resumo em Inglês:

Abstract Artificial insemination with cooled semen is the most common practice in rabbit farms and any improvement on it helps to increase the efficiency and productivity of rabbit meat farms. Therefore, the aim of this study was to assess whether different cryoprotectant agents (CPA) as glycerol, N, N-Dimethylformamide (DMF) and N-Methyl-2-Pyrrolidone (NMP) can improve cooled rabbit sperm quality stored at 4ºC and 16ºC. Sperm samples were diluted with INRA 96® (Extender A), INRA 96® with 6% glycerol (Extender B) or 6% DMF (Extender C) or 6% NMP (Extender D) respectively and stored at 4ºC and 16ºC. Samples were then analysed at 4, 24, 48 and 72 hours after refrigeration by integrated sperm analysis system (ISAS®), eosin-nigrosin stain (vitality), hypo-osmotic swelling test (HOS test) and acrosome integrity test. Extender C showed higher percentage of motility, vitality and HOS test than extender B and D (p<0.05). Whereas sperm quality decreased over time (p<0.05), data showed that the addition of DMF kept the motility and sperm plasma membrane integrity after 24 hours of storage better than other diluents. These results suggest that the addition of DMF to INRA 96® exerts a protective effect on the membrane of spermatozoa improving seminal quality.

Resumo em Inglês:

Abstract The objective of this study was to investigate the need of seminal plasma removal for short-term cooling of buck semen in soybean lecithin (SL) based extender. Each pool was divided equally, and one half was subjected to centrifugation to remove seminal plasma (SP-), while the other half remained with seminal plasma (SP+). Then, both SP+ and SP- samples were diluted in two SL extenders (extender A = 1% SL; extender B = 2% SL), cooled to 5ºC and stored for 48 hours. The sperm kinetics, evaluated by CASA, and plasma membrane integrity (PMI), acrosomal integrity (ACI) and high mitochondrial membrane potential (HMMP), evaluated by epifluorescence microscopy, were determined within five minutes after reaching 5°C (T0), as well as after 24 (T24) and 48 (T48) hours of storage. Interactions (seminal plasma vs. extender vs. time;) were observed for all variables assessed. Total and progressive motility and other variables of sperm kinetics decreased after 24 hours of cooling in the SP+ group, and after 48 hours of storage, these same variables were lower in SP+/B compared to SP-/B groups. Furthermore, SP+ reduced PMI (extender B, T48), HMMP (A and B extenders, T48) and ACI (extender A, T0) compared to SP- samples. The interactions between seminal plasma and soybean lecithin phospholipids seemed to occur in a time-dependent manner. It was concluded that the removal of seminal plasma improves the quality of goat semen that was cooled in a soybean lecithin-based extender, especially when using 2% soybean lecithin.

Resumo em Inglês:

Abstract The Saanen goat breed has been widely explored in breeding programmes; however, there are few reports about the breed’s genetic and molecular composition. Thus, this study aimed to characterize the proteomic profile of spermatozoa from Saanen breeding goats. Five breeding animals with proven fertility were selected, the spermatozoa were collected, and the protein was extracted. Subsequently, the proteins were separated and analysed by two-dimensional electrophoresis and mass spectrometry; the proteins were then identified with the SwissProt database. A total of 31 proteins involved in reproduction were identified, including binding proteins on spermatozoa for fusion with the egg, acrosomal membrane proteins, metabolic enzymes, heat shock proteins, cytoskeletal proteins and spermatozoa motility proteins. The characterization of such proteins clarifies the molecular mechanisms of spermatogenesis and the modifications that ensure the success of fertilization.

Resumo em Inglês:

Abstract The aim of this study was to evaluate the long-term effects of recombinant bovine somatotropin (rbST) on follicle population and ovulatory follicle development in non-lactating dairy cows. Twenty-one Jersey cows were allocated in rbST (n=11) or control (n=10) groups. On day -60, cows in rbST group received 500 mg of somatotropin (s.c. Lactotropin®, Elanco). On day 0, control and rbST cows received an intravaginal progesterone-releasing device (1.9 g, CIDR®, Zoetis) and GnRH (100 mg, IM, Factrel®, Zoetis). On day 8, cows received PGF2α (25 mg, IM, Lutalyse®, Zoetis) and the CIDR® was removed. Twelve hours after device removal (D8), serum, follicular fluid and granulosa cells samples were collected. Serum and follicular concentration of estradiol (E2) and progesterone (P4) were analyzed. Total RNA was extracted from granulosa cells to measure gene expression of LHCGR, STAR, HSD-3B1, CYP11A1, CYP19A1, CYP17A1, IGFR and PAPPA by real-time PCR. Ultrasonography was performed on days -60, -53, -46, -14, -7, 0 and 8 for antral follicle count. Results were analyzed by repeated measures ANOVA and t-test. There was no effect of rbST treatment on the number of follicles during the 60 days period, as well as no effect on serum and follicular fluid E2 and follicular fluid P4 at the moment of follicle aspiration. There was a reduction in PAPPA (P = 0.006), CYP11A1 (P = 0.04) and CYP19A1 (P = 0.002) mRNA levels in granulosa cells of the pre-ovulatory follicle of rbST treated cows. In conclusion, a single dose of rbST did not have long-term effects on antral follicle population, serum and follicular E2/P4 concentrations in non-lactating dairy cows. Despite that, rbST injection decreased granulosa cell expression of genes related to steroidogenesis in the pre-ovulatory follicle.

Resumo em Inglês:

Abstract Ultrasonographic examination of pregnant ewes can enable the identification of perinatal abnormalities and establish prenatal assistance responsible for minimizing morbidity and perinatal mortality. Therefore, we aimed to evaluate the feasibility of a fetal biometric analysis by ultrasonography to predict neonatal vitality and lamb growth during the first month of life. A longitudinal study was conducted with 13 healthy ewes, subjected to ultrasonographic examination every 15 days from 60th day of pregnancy until lambing, evaluating thoracic diameter, abdominal diameter, biparietal diameter, humerus, femur and placentome length. At birth, 22 lambs were assessed through Apgar score at 5 minutes and after 1 hour. Measurement of body weight was also carried out immediately at birth and weekly during 30 days after birth. Thoracic diameter showed a significant increase between 91-105 days and 121-135 days. Conversely, abdominal diameter had a progressive growth until 106-120 days, and then, a steady development was observed. Biparietal diameter showed progressive growth only towards days 91 and 105. For the humerus length, we verified a significant increase between 106-120 days and 121-135 days, remaining unaltered onwards; while femur length continued to grow until lambing. The linear regression analysis between birth weight and biparietal diameter at 60-75 days was high (R2=0.96; P<0.0001; coefficient of variability of 3.3%). In conclusion, ultrasonographic analysis of fetal biparietal diameter at mid-pregnancy can be used as a predictor of lamb weight at birth. Moreover, assessment of femur length at final pregnancy can be employed for fetal and neonatal development estimation.

Resumo em Inglês:

Abstract Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis (PTB), disease that causes a syndrome of bad nutrient absorption, weight loss and eventually death. The intestine is the main target organ where the infection develops; however, there is evidence of infection by MAP in extra-intestine sites of sheep, including mesenteric nodes and semen. The aim of the study was to identify the presence of MAP in reproductive tissue and semen of infected Pelibuey rams in clinical state of PTB. Seven rams were used in clinical PTB state and a non-infected ram by MAP of the Pelibuey breed, confirmed by serology, nPCR and bacteriological culture, with average weight and age of 57.23 ± 1.73 kg and 2.91 ± 0.17 years, respectively. The presence of MAP was identified in different tissue samples: spleen (1/7, 14.3% and 2/7, 28.6%), small intestine (3/7, 42.9% and 4/7, 57.1%) and mesenteric lymph nodes (3/7, 42.9% and 3/7, 42.9%), with nPCR and culture, respectively. It was also identified in epididymis tissue (1/7, 14.3%), Cowper gland (2/7, 28.6%) and prostate (1/7, 14.3%), using nPCR, although without detection in culture. It was identified in testicular tissue in 42.8% (3/7; culture or nPCR technique), but in 28.6% (2/7) with both techniques. Finally, the presence of MAP was identified in 42.9% (3/7) of semen samples with nPCR; however, it was not detected through culture. In conclusion, the presence of MAP was identified in lymphatic, digestive tissue, and semen; the presence of MAP was reported for the first time in epididymis, Cowper gland, prostate and testicles of infected Pelibuey rams.

Resumo em Inglês:

Abstract We aimed to evaluate the effects of L-arginine (L-arg) in the quality of in vitro heparin-induced capacitation of cryopreserved bovine spermatozoa and its effects on IVP. The experimental groups were: Control 0 hour without pre-capacitation, and groups of sperm capacitated for 30 min in the absence of COC with heparin (Control 30 min), with 1 mM L-arg and with 1 mM L-arg + heparin. The capacitation pattern was evaluated by chlortetracycline assay and the integrity of the plasma membrane (PM) and acrosome membrane (AM) by the association of Hoescht 33342 and propidium iodide. Further, we assess the sperm quality by the rate of in vitro blastocysts production. Treatment with 1 mM L-arg + heparin increased the percentage of capacitated sperm when compared to Control 0 hour and the treatment with heparin (61.1 vs. 18.2 and 47.0%, respectively, P<0.05). The addition of 1 mM L-arg to the medium has capacitated the spermatozoa (26.2 ± 3.8) but was less effective than heparin (47.0 ± 4.0) (P<0.05). There was no difference in the percentage of sperm with intact PM between treatments when compared to Control 0 hour (P>0.05). The group capacitated with 1 mM L-arg + heparin for 30 min increased the blastocyst rate compared to Control IVF (53.7 vs. 40.8%, P<0.05). We conclude that the addition of L-arg with heparin increases the number of capacitated spermatozoa in vitro with 30 min of pre-incubation in the absence of COC not altering the integrity of plasma and acrosomal membrane. This treatment in the absence of COC was the most effective method for blastocysts production, and the method of pre-incubation could be used to assess the role of other substances in the sperm capacitation and its effect on IVP.