SUBPOPULATION STRUCTURE AND CHANGES AFTER CRYOPRESERVATION OF SPERMS FROM HIGH AND LOW FERTILITY WATER BUFFALO / ESTRUCTURA DE LAS SUBPOBLACIONES Y CAMBIOS DESPUÉS DE LA CRIOPRESERVACIÓN ESPERMÁTICA EN BÚFALOS DE AGUA CON ALTA Y BAJA FERTILIDAD

The aim of this study was to identify the sperm subpopulation structure in buffalo bulls with high and low fertility and to determine how sperm subpopulations change after semen cryopreservation. Semen was obtained from four bulls with high fertility (HF) and four bulls with low fertility (LF) and was cryopreserved. A total of 64 ejaculates were assessed for their sperm kinematics using computer assisted sperm analyzer (CASA). Ward’s Hierarchical Dendogram and K-Means clustering method were used to identify the subpopulations. In experiment 1, two significantly different (P≤0.05) sperm subpopulations were observed: Subpopulation 1 (SP1): sperms travel longer distances most rapidly and progressively, and Subpopulation 2 (SP2): sperms travel shorter distances slower but highly progressive. A higher percentage of SP1 was found in HF bulls (47.27); whereas, a higher percentage of SP2 was found in LF bulls (54.89). A low negative relationship (r=-0.18) was observed for the fertility level and sperm subpopulation structure. This implies that sperms that travel longer distances most rapidly and progressively (SP1) are most likely associated to high fertility, while sperms that travel shorter distances slower but highly progressive (SP2) are associated with low fertility. In experiment 2, based on the change in SP1 after cryopreservation, significantly higher sperm survival was observed in samples from HF bulls (29.97) as compared to those from LF bulls (31.64). During post thaw, there were more SP1 sperms in samples from HF bulls (27.52) than in those from LF bulls (26.74). Thus, semen containing higher proportion of SP1 sperms are more resistant to cryopreservation and have greater chances of obtaining high fertility. Overall, the identification of sperm heterogeneity in water buffaloes can be associated to sperm survival after cryopreservation and fertility. RESUMENEl propósito de este estudio fue identificar la estructura de las subpoblaciones espermáticas en toros bufalinos con alta y baja fertilidad y determinar los cambios luego de la criopreservación. El semen se obtuvo de cuatro búfalos con alta fertilidad (HF) y cuatro con baja fertilidad (LF) y fue criopreservado. Un total de 64 eyaculados fueron evaluados para parámetros cinéticos usando un analizador espermático asistido por computadora (CASA). El método de dendograma jerárquico de Ward y el método K-means fueron utilizados para identificar las subpoblaciones. En el experimento 1, dos subpoblaciones espermáticas estadísticamente diferentes (p<0.05) fueron observadas: Subpoblación 1 (SP1): espermatozoides que viajan largas distancias más rápida y progresivamente, y la Subpoblación 2 (SP2): espermatozoides que viajan distancias cortas de forma lenta pero muy progresivamente. Un mayor porcentaje de SP1 fue encontrado en los búfalos HF (47,27); mientras que un mayor porcentaje de SP2 fue encontrado en los búfalos LF (54.89). Una relación baja pero negativa (r = -0,18) fue observada para el nivel de fertilidad y la estructura de la subpoblación espermática. Esto implica que los espermatozoides que viajan largas distancias más rápida y progresivamente (SP1) están más asociados a la alta fertilidad, mientras que los que viajan distancias cortas más lento y con alta progresividad (SP2) están asociados a una baja fertilidad. En el experimento 2, en base a los cambios en SP1 luego de la criopreservación, un mayor porcentaje de espermatozoides permaneció en esta subpoblación en los búfalos HF (27,52) que en los LF (26,74). Por lo tanto, semen con una alta proporción de espermatozoides dentro de SP1 son más resistentes a la criopreservación y tienen mayor probabilidad de obtener una mayor fertilidad. En general, la identificación de la heterogeneidad espermática en búfalos de agua puede ser asociada a la sobrevivencia luego de la criopreservación y a la fertilidad

Influence of Ovarian Follicle Sizes and Estrous Signs on Pregnancy Following Progesterone-Based Fixed Time Artificial Insemination in Water Buffaloes

The objectives of the present study were to elucidate the importance of follicle sizes and estrous signs during Controlled Internal Drug Release-Synch-human Chorionic Gonadotropin (CIDR-Synch-hCG) protocol for Fixed Time Artificial Insemination (FTAI) and to evaluate their association with pregnancy in water buffaloes. Data from riverine buffaloes (n = 207) under the CIDR-Synch-hCG protocol were analyzed. Buffaloes were administered with Gonadotropin-Releasing Hormone (GnRH) with insertion of CIDR on Day 0. Prostaglandin (PGF2α) was given on Day 7 with the removal of CIDR. hCG was given on Day 9, and AI was performed on Day 10. Follicle measurements by ultrasonography were done on Days 0, 7, and 10, and follicle sizes on those days were categorized into I, II, and III. Estrus signs were taken on the day of AI. The pregnancy diagnosis was done on Day 30-35 post-AI. The average size of follicles in Category III is significantly higher than those of Categories I and II, regardless of the Days of the protocol. Pregnancy is significantly (P&lt;0.001) associated with Pre-Ovulatory Follicle (POF) size and uterine tonicity on the Day of AI but not with follicle sizes on Days 0 and 7, nor with mucus discharge discharge (P&gt;0.05). The overall pregnancy rate is 44.44% while performing AI with POF size ≥12.0 mm increased the probability of pregnancy rate to 56.25%. In conclusion, the present study demonstrated a follicle size-based CIDR-Synch-hCG protocol providing new fertility indicators to improve FTAI efficiency in buffaloes with huge application in other livestock species

Effects of isolation method and pre-treatment with ethylene glycol or raffinose before vitrification on in vitro viability of mouse preantral follicles

Effects of isolation and vitrification protocols on follicular survival after warming were examined. Mouse preantral follicles enzymatically or mechanically isolated from ovaries of 12-day-old mice were exposed either to 2 M ethylene glycol (EG) for 2 or 5 min, or to ascending concentrations (0.15 then 0.3 M) of raffinose for 2 or 5 min each (2-2 and 5-5 min). They were then exposed to a vitrification solution (VS) composed of 6 M EG and 0.3 M raffinose for 0.5, 1, or 2 min before vitrification. Mechanically isolated follicles showed higher survival than enzymatically isolated follicles, regardless of periods of exposure to EG or raffinose and subsequent exposure to VS. After 10 days of culture, follicular growth and maturational ability of oocytes derived from vitrified follicles exposed to 2 M EG for 5 min and to VS for 1 min were higher than those from follicles exposed to raffinose solutions for 2-2 min and to VS for 1 min. Histological evaluation revealed that exposure of preantral follicles to raffinose solutions caused cytoplasmic vacuolation in granulosa cells which could be due to cellular shrinkage during dehydration; whereas, exposure to 2 M EG induced morphological alterations in follicles only to a lesser extent