Evaluation of the Freezability of the Bovine Epididymis Tail Sperm with the Addition of Antioxidants.

BACKGROUND: The cryopreservation and recovery of epididymis tail sperm is an important biotechnology dependent on the composition of the freezing medium. OBJETIVE: To evaluate the effect of melatonin, added to commercial freezing medium extender, on the kinetics and viability of bovine epididymis tail sperm. MATERIAL AND METHODS: Five routines were performed, each consisting of eight epididymis and the structures were sliced onto a glass plate containing a commercial diluting medium for Botubov. The samples were divided into four groups, with 80 x 106 spermatozoa per mL. Group 1: samples diluted in Botubov. Group 2: samples centrifuged (600 g, 10 min), and the pellet re-suspended in Botubov. Group 3, samples diluted in Botubov containing 100 pM melatonin. Group 4: samples centrifuged (600 g, 10 min) and the pellet resuspended in Botubov with 100 pM melatonin. The samples were transferred to 0.5 mL straws at 40 x 106 viable spermatozoa, stabilized at 5º C for 4 h, transferred to liquid nitrogen vapour for 20 min, dipped in liquid nitrogen and stored in a cryogenic cylinder. After thawing (46ºC, 15s), sperm kinetics and viability parameters were evaluated. RESULTS: There was no difference in the parameters of total motility (MT, %), progressive motility (MP, %), progressive linear velocity (VSL, µm/s), curvilinear velocity (VCL, µm/s), linearity (LIN, %), spermatozoa with rapid movement (RAP, %) and level of intact plasma membranes and acrosome (IPMA, %) among the groups studied. However, a difference was observed between the routines performed. CONCLUSION: The protocol for freezing bovine epididymis tail sperm is applicable; however, there is an influence of the epididymis used, for the best efficacy of this biotechnology.

Gene Expression of Fresh and Frozen/Thawed Canine Embryos.

BACKGROUND: Canine embryo cryopreservation and subsequent transfer are relevant in the use of reproductive technologies. OBJECTIVE: The purpose of this study is the identification and quantification of the gene expression BAX and Bcl2, AQP3, Na+/K+ ATPase alpha-1 and beta-1 and LIFr in canine embryos obtained in vivo and after freezing. MATERIALS AND METHODS: For the collection of embryos, the bitches were identified at pro-estrous until the detection of 80-90% superficial cells. After that, they were artificially inseminated with fresh semen. The embryos were collected after ovariohysterectomy. RNA was extracted and amplified, and embryos were randomly distributed into fresh (Fr) and frozen/thawed (Ft) groups. RESULTS: Eighteen blastocysts were collected from three bitches. Genes BAX, AQP3 and LIFr did not differ among the studied groups. CONCLUSION: We suggest, through these results, that the genes BAX, Bcl2, AQP3, Na + / K + ATPase alpha-1 and beta-1 and LIFr were expressed in canine blastocysts collected in vivo and after slow freezing cryopreservation.

Pentoxifylline effects on capacitation and fertility of stallion epididymal sperm.

The aims of this study were to determinate whether pentoxifylline (PTX) increases the motion parameters of fresh and frozen-thawed equine epididymal spermatozoa, to evaluate the tyrosine phosphorylation of frozen-thawed epididymal sperm in the presence of PTX and to determine whether the PTX-treatment of stallion epididymal sperm prior to freezing improves the fertility response of mares to a reduced number of spermatozoa per insemination dose. Fifty epididymis were flushed with a skim milk based extender with or without PTX. The pre-treatment with PTX enhanced the sperm motility after being harvested (P<0.05); however the freeze-thaw process did not alter the sperm kinematics between control and treated samples (P>0.05). Plasma membrane integrity did not differ between control and PTX group after recovery and after thawing (P>0.05), as observed in tyrosine phosphorylation, which the PTX treatment did not alter the percentage of tail-associated immunofluorescence of cryopreserved epididymal sperm (P>0.05). For the fertility trial, different insemination groups were tested: 800×106 epididymal sperm (C800); 100×106 epididymal sperm (C100); 100×106 epididymal sperm recovered in an extender containing PTX (PTX100). The conception rates for C800; C100 and PTX100 were 68.7% (11/16); 31.5% (5/16) and 50% (8/16), respectively. The conception rate did not differ among groups (P>0.05), however, a low number of animals was used in this study. A trend toward significance (P=0.07) was observed between C800 and C100 groups. In conclusion, PTX has no deleterious effect on sperm motility, viability and capacitation of cryopreserved stallion epididymal sperm. The conventional artificial insemination with 100×106 sperm recovered with PTX ensures acceptable conception rates and maximize the limited number of doses of cryopreserved stallion epididymal sperm.

Effect of Temporary Meiotic Attenuation of Oocytes with Butyrolactone I and Roscovitine in Resistance to Bovine Embryos on Vitrification.

This study aimed to produce in vitro bovine embryos by the addition of two drugs, which is responsible for oocyte meiosis inhibition: roscovitine (ROS) and butyrolactone I (BL-I). Oocytes were recovered from slaughtered cows and matured in a commercial medium and maintained in a 5% CO2 atmosphere. Oocytes were maintained for 6 h in an in vitro maturation (IVM) medium containing ROS (12.5 µm), BL-I (50 µm) and association of drugs (ROS 6.25 µm and BL-I 25 µm). Oocytes were cultured for 18 h in an agent-free medium for the resumption of meiosis. After 24 h of maturation, oocytes were inseminated in the commercial in vitro fertilization (IVF) medium. Presumptive zygotes were cultured in SOFaa medium in a 5% CO2 atmosphere. On day 3, rate of cleavage was evaluated and on days 6 and 7, rate of blastocyst formation. BL-I and its association with the ROS increased the rates of cleavage and blastocyst formation (p < 0.05). The ROS alone was inefficient, impairing embryonic development, with low rates of blastocyst formation when compared to the control group and other treatments (p < 0.05). The embryos from BL-I and ROS+BL-I groups presented higher number of cells and lower rates of cellular apoptosis compared to other groups, either for the fresh or for post-thawing embryos. Embryos from ROS+BL-I group showed to be more resistant to the vitrification process, presenting a higher rate of embryonic re-expansion (p < 0.05). In conclusion, block of meiosis using BL-I or its association with ROS increased the rate of blastocyst formation, and the association of ROS+BL-I resulted in a better resistance to the embryo cryopreservation process.

Use of cholesterol-loaded cyclodextrin: an alternative for bad cooler stallions.

During the cooling process, sperm may suffer irreversible damage that compromises the fertility rate. Incorporating cholesterol-loaded cyclodextrin (CLC) represents a strategy to increase sperm resistance at low temperatures; however, high levels of cholesterol in the cell membrane can interfere with sperm capacitation. The goals of this study were to determine the CLC concentration and cooling temperature that produce optimal kinetic parameters and viability of sperm from stallions identified as bad coolers (BCs) and good coolers (GCs), as well as the effect of adding CLC on the occurrence of the acrosome reaction (ACR) and on the fertility rate of cooled sperm. In experiment 1, each ejaculate was divided into four groups: Control and treated with 1 (CLC-1), 1.5 (CLC-1.5), or 2 mg (CLC-2) of CLC/120 × 10(6) sperm and cooled for 48 hours at 5 °C. In experiment 2, each ejaculate was divided into four groups: Control and CLC-1.5 cooled at 15 °C or 5 °C for 24 hours. For experiment 3, GC and BC stallions were used, and the ejaculates were divided into control and CLC-1.5 cooled at 5 °C for 48 hours. According to experiment, the sperm kinetics (SK) and plasma membrane integrity (PMI) were analyzed before and after 24 and 48 hours of cooling. In experiment 4, the ejaculates were divided into four groups: Control and CLC-1.5 maintained at room temperature or cooled at 5 °C for 24 hours. Each group was incubated with ionophore calcium at 37 °C for 3 hours. The incidence of ACR was analyzed before and after 1, 2, and 3 hours of incubation. For experiment 5, two cycles of 10 mares for a GC stallion and two cycles of 25 for a BC stallion were used. The inseminations were performed with control and CLC-1.5 groups cooled at 5 °C for 24 hours. According to results, all groups treated with CLC exhibited higher PMI compared with controls, and CLC-1.5 and CLC-2 exhibited the best SK results. The cooling temperature of 5 °C was superior to 15 °C when the sperm was treated with CLC. The GC and BC stallions benefited from the CLC-1.5 treatment, but the BCs were more evident, which presented greatly increased PMI and SK. There was a delay in capacitation of at least 3 hours for the fresh sperm and at least 1 hour for cooled sperm supplemented with CLC-1.5. After adding CLC-1.5, the fertility of BC stallion significantly increased, but that of the GC was not altered. Thus, incorporating CLC is an effective technique to cool equine semen, although it is indicated mainly for BC stallions.

Cryopreservation and fertility of ejaculated and epididymal stallion sperm.

The cryopreservation of epididymal sperm is important to preserve genetic material from valuable deceased males. This study evaluated the viability of sperm samples from eight stallions under three conditions: (1) collected using an artificial vagina (EJ-0h), (2) recovered from the epididymal cauda immediately after orchiectomy (EP-0h), and (3) recovered from the epididymal cauda after 24h of storage at 5°C (EP-24h). To obtain EJ-0h sperm, two ejaculates were collected from each stallion. After 1 week, the stallions were submitted to bilateral orchiectomy, and one of the removed epididymides was flushed to obtain EP-0h sperm. The contralateral epididymis was stored at 5°C for 24h before being flushed to obtain EP-24h sperm. The sperm samples were analyzed at three different times: immediately after sperm recovery, after dilution in the freezing extender, and post-thawing. A fertility trial was performed using 39 estrous cycles. After ovulation induction with 1mg of deslorelin acetate (i.m.), mares were inseminated with 800×10(6) sperm. The total number of sperm recovered was 7.8±4.7×10(9) for EJ-0h sperm, 12.9±9.2×10(9) for EP-0h sperm and 12.0±8.0×10(9) for EP-24h sperm. The sperm motility, evaluated by total motility, progressive motility and the percentage of rapid cells, was similar among the samples before and after freezing (P>0.05). However, the plasma membrane integrity was different between EJ-0h and EP-0h pre-freezing and between EJ-0h and EP-24h post-thawing (P<0.05). The conception rates were similar between groups inseminated with sperm recovered from the epididymal cauda immediately after orchiectomy (EP-0h), after 24h of storage at 5°C of the epididymal cauda (EP-24h) and with ejaculated sperm (EJ-0h) (P>0.05). In conclusion, the viability and fertility of cauda epididymal sperm are similar to those of ejaculated sperm.