Preface

Journal of Buffalo Science, Volume 1 Number 1, Prefac

Effect of Royal Jelly on the Fertilizing Ability of Buffalo Spermatozoa In Vitro

The aim of the present study was to assess the effect of addition of Royal jelly in presence of heparin on buffalo (Bubalus Bubalis) sperm motility, acrosome reaction and in vitro fertilization (IVF) of buffalo oocytes. Frozen buffalo spermatozoa from five bulls were thawed and motile fraction was obtained by swim up technique. The spermatozoa were washed, treated with100 µg/ml heparin, and then exposed to 0.4% Royal Jelly (RJ) for 3 h. Sperm motility, acrosomal integrity and fertilization rate of matured oocytes were assessed at 1, 2 and 3 h. The percentages of sperm motility, intact acrosome and fertilization rate of matured oocytes were higher (P<0.05) in 0.4% RJ compared to that in the control. After 2 h of incubation the percentage of motility, intact acrosome of spermatozoa and fertilization rate of matured oocytes, respectively, were 93.6 %, 77.6% and 72.6% in 0.4% RJ. These results suggest that treating buffalo sperm with 0.4% RJ in combination with heparin is effective not only to induce sperm acrosome reaction but also is effective for in vitro fertilizing capacity of the cryopreserved buffalo spermatozoa

Efectos de la incubación in vitro con jalea real sobre las características del semen bovino descongelado

Cryopreserved bovine semen is generally considered to have lower fertility compared to fresh semen. The reduction arises from both a lower post–thaw viability and a possible sublethal dysfunction of the surviving sperm population. The present study was conducted to observe the effect of royal jelly (RJ) on motility, viability and acrosome integrity of spermatozoa during post–thaw incubation. Frozen–thawed semen samples were washed and incubated at 37°C in Tris buffer containing 0.1, 0.2, 0.3, 0.4, 0.5% RJ or none (control). Sperm motility, viability and acrosomal integrity were assessed at 0, 0.5, 1, 1.5 and 2 h. The percentages of sperm motility, viability and intact acrosome were higher in Tris buffer containing 0.4% RJ compared to control (p < 0.05). After 2 h of incubation the percentages of motility, viability and intact acrosome of spermatozoa, respectively, were 52.3; 52.5 and 19.8% in 0.4% RJ containing buffer. Results indicate that the addition of 0.4% RJ in the incubation media was able to maintain better quality and longevity of spermatozoa. Royal jelly may be used as a semen extender to improve sperm quality and fertility.El semen bovino criopreservado generalmente es considerado de menor poder fertilizante comparado con el semen fresco. Tal reducción abarca tanto una más baja viabilidad post–descongelado como una posible disfunción subletal de la población de espermatozoides sobrevivientes. El presente estudio fue realizado para determinar el efecto de la jalea real (JR) sobre la motilidad, viabilidad e integridad acrosomal de espermatozoides durante la etapa de incubación post–descongelado. Las muestras de semen congelado–descongelado se lavaron e incubaron a 37°C en buffer Tris conteniendo JR en proporción de 0,1; 0,2; 0,3; 0,4; 0,5% o ninguna (control). La motilidad y la viabilidad espermáticas, así como la integridad acrosomal, fueron evaluadas a las 0; 0,5; 1; 1,5 y 2 h. Comparados con los controles, los porcentajes de motilidad, viabilidad e integridad acrosomal fueron más altos en los espermatozoides incubados en Tris conteniendo 0,4% de JR (p < 0,05). Después de 2 h de incubación, los porcentajes de motilidad, viabilidad e integridad acrosomal de los espermatozoides fueron respectivamente de 52,3; 52,5 y 19,8% en el buffer que contenía JR al 0,4%. Los resultados indican que la adición de 0,4% de JR en los medios de incubación fue capaz de mantener la buena calidad y longevidad de los espermatozoides. La jalea real puede ser usada como aditivo seminal para mejorar la viabilidad y fertilidad de los espermatozoides

In Vito Fertilization in Buffaloes: A Review

This is the review of original data concerning the effect of some factors on oocyte development in vitro of buffaloes. In vitro fertilization is a multi - step process: oocytes maturation, fertilization and embryo culture. In vitro fertilization is strongly influenced by events occurring during oocyte maturation, fertilization and the subsequent development of the fertilized oocytes. With the advancement of IVF procedures, variability in developmental rate and viability of in vitro produced buffalo embryos so, improving the efficiency and identifying the sources of variations between IVF systems are more important when routinely producing blastocysts from individuals of high genetic merits. Also, the development of specific culture regimes capable of supporting in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) to the blastocyst stage is highly desirable in breeding systems. This paper discusses the technical aspects of the procedures involved in in vitro fertilization of buffaloes

Evaluations of Ovarian and Luteal Blood Flow Waveform Patterns in Buffalos Subjected to OvSynch Protocol in Cold and Hot Seasons

This current study aimed to determine ovarian and luteal blood flow waveform patterns in buffalos synchronized using OvSynch protocol in cold and hot seasons. Six cyclic buffalo cows aged 6±0.5 years old, having a weight of 400 ± 50 kg, were scanned daily along three successive estrous cycles transrectally by Doppler ultrasonography to evaluate the normal ovarian hemodynamic during the normal spontaneous ovulation and before the start of experiments. Buffaloes were synchronized with gonadotropin[GnRH] –prostaglandin[P] –gonadotropin (GPG) protocol in which animals received 10μg of GnRH on day ??, 0.250μg of PGF2α on day 7, and another dose of 10μg of GnRH was administered 48h after the PGF2α injection. Blood sampling and ovarian ultrasound examinations (color and spectral Doppler modes) were conducted on the day of the estrous and luteal phases. Results revealed that peak systolic velocity waveform (PSV) was significantly (P<0.05) increased in the cold season compared to the hot season. The Luteal blood flow after the end of OvSynch protocol on days (5,7,9, and 11) was significantly increased in the cold season than that in the hot one. The serum levels of estradiol (E2) and nitric oxide (NO) after the second GnRH injection in the OvSynch protocol were significantly (P<0.05) elevated in the cold season as compared to the hot one. Moreover, the progesterone (P4) levels had risen in OvSynch-treated buffaloes on days 5,7,9, and 11 of the cycle in the cold season compared to the hot one. Conclusion: In the cold season, ovarian hemodynamics was significantly improved compared to the hot one; this may influence the reproductive efficiency of buffaloes. Further studies were needed to prove it

Preparation of cyclodextrin nanoparticles and evaluation of its effect on the capacitation of bovine spermatozoa used in the in vitro fertilization

This study was conducted to produce nanosized cyclodextrin (NCD) and assess its effect on bovine spermatozoa during In vitro fertilization (IVF) to optimize the capacitation media for successful IVF. Therefore, Four cyclodextrin formulations were prepared and characterized. Data analysis revealed the best formula (F2) showed a smallest particle size (15 nm), zeta potential (-37 mv), and higher yield percentages (95%) was selected for spem capacitation. Motile spermatozoa were separated from frozen-thawed semen by a swim-up procedure and capacitated in IVF-TALP medium with different formulae of NCD or CD or without treatments (control) and incubated for 3hours(hr) at 38°C and evaluated every one (hr) interval. Data analysis revealed that the formulation of cyclodextrin nanoparticles (F2) after (2hr) incubation in the media gave best effect on sperm capacitation and acrosme reaction (AR) and effect of sperm treated with NCD on fertilization rate was evaluated. The results showed that the proportion of Oocytes fertilized was increased significantly in F2 (60%) than in the control (35%), and cyclodextrin group (50%) groups (p<0.05). It could be inferred from this investigation that cyclodextrin nanoparticles can be used for biomedical interventions in bovine spermatozoa. NCD improve sperm motility, viability, and (AR), also fertilization rate of sperm treated with NCD increase. So NCD gave positive effect on sperm functions during IVF.

Correlation between Uterine Hemodynamics, Sex Steroid Hormone Concentrations, and Enzymatic Antioxidant Levels in Postpartum Buffaloes

The present investigation aimed to evaluate uterine hemodynamics in six multiparous postpartum buffaloes and their relationship with sex steroid hormone concentrations and enzymatic antioxidant levels. The buffaloes were examined by transrectal Doppler ultrasonography to record the vascular perfusion in uterine arteries of both ipsilateral and contralateral ones. All Doppler indices such as peak (PV) endpoints (EV) of velocity, peak systolic velocity (PSV), blood flow volume (BFV), resistance (RI), and pulsatility index (PI) were recorded from 1st to 6th postpartum. The blood samples were collected starting from 1st-week post-calving and every week thereafter following each ultrasound Doppler examination for assay of steroid hormones (progesterone and estradiol) and antioxidant (superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase, CAT) were measured. PV of the ipsilateral previously gravid arteries showed a linear pattern of significant (P=0.001) decline from the 1st week after parturition till the 6th week. This decline was also associated with a linear decrease in EV from the 1st week till the 6th week after birth. While contralateral PV and EV are not significantly changed throughout weeks after parturition. The levels of SOD and CAT are significantly elevated at 1st week postpartum compared to the 6th week after parturition. In contrast, the GPx levels did not reveal any significant differences during the puerperal period. Estradiol and progesterone declined from 1st to 5th week after parturition. PV of the ipsilateral uterine artery had a significant (P≤0.05) positive correlation with BFV (r=0.49), estradiol 17- α(r=0.98) and progesterone (r=0.85). The same parameter showed a statically (P≤ 0.001) positive correlation with SOD (r=0.87) and CAT (r=0.92). While, Ipsilateral uterine RI showed a significant (P≤0.05) negative correlation with PV(r=-0.85), BFR ((r=-0.62), estradiol 17- α(r=-0.52), and progesterone (r=-0.88), in addition, RI also correlated negatively with both SOD (r=-0.57) and CAT (r=-0.63). Progesterone and estrogen levels are strongly correlated with SOD and CAT. The uterine hemodynamics in buffaloes is affected by the day of the postpartum period. SOD and CAT antioxidants recorded herein, except GPx, increase in the 1st weeks of calving and are affected by the day of the postpartum period

Preparation of progesterone nanoparticles and evaluation of its effect on the capacitation of Bovine spermatozoa used in the in Vitro Fertilization

Progesterone (P) has been reported to affect several sperm functions especially capacitation and acrosome reaction. The main problem of (P) is its low aqueous solubility. So formulation of progesterone nanoparticles (PN) will enhance its solubility. This study was conducted to produce nanosized progesterone (NP) and assess its biocompatibility. Therefore, nine progesterone formulations were prepared and characterized. Data analysis revealed only one formula of P showed nanosized particle (1-100 nm) with an average particle size (95±5 nm), and spherical shape as seen by Transmission Electron Microscope(TEM). Motile spermatozoa were separated from frozen-thawed semen by a swim-up procedure and capacitated in IVF-TALP medium with NP or P or without treatments (control) and incubated for 3h at 38°C and evaluated every 1 hour (h) interval. Ovarian oocytes were matured and fertilized in vitro with frozen-thawed bull sperm capacitated in vitro with NP or P or control (without NP, P) and incubated at 39C in 5% CO2 incubator for 24h and then examined for evidence of fertilization. In conclusion, this study demonstrates that nanosized progesterone is highly efficient for sperm capacitation. In addition to the use of nanosized progesterone in sperm capacitation produces more fertilized oocytes than the progesterone after In Vitro Fertilization (IVF)

In vitro production of buffalo embryos from stepwise vitrified immature oocytes

This study was conducted to produce buffalo embryos in vitro from stepwise vitrified immature oocytes. Cumulus oocyte complexes (COCs) were obtained from the ovaries of slaughtered buffalo and were collected from the local abattoir. Selected COCs were exposed to a vitrification solution consisting of 40% ethylene glycol (EG) plus 0.3 M trehalose and 20% polyvinyl pyrrolidone (PVP) for 1 min and loaded in 0.25 ml plastic mini-straws containing 100 µl of 10% sucrose. The loaded cryostraws were cryopreserved by stepwise vitrification and were stored in liquid nitrogen for 4 to 6 months. Data analysis revealed a high percentage of post-thawing morphologically normal immature oocytes (80.7%) with a low percentage of damaged oocytes. There were no significant differences in the maturation (82.1%), cleavage (47.6%) and buffalo embryo development (15.4%) produced by the stepwise vitrified immature oocytes in comparison to the three observations in fresh oocytes (88.3%, 50.4% and 19.4%, respectively, p<0.05)

Successful cryopreservation of buffalo ovaries using in situ oocyte cryopreservation

To improve the efficiency and efficacy of cryopreservation of ovaries, we developed a new method termed in situ oocyte (ISO) cryopreservation. ISO cryopreservation is a multistep procedure that involves aspiration of follicular fluid and then perfusion of antral follicles and diffusion of whole buffalo ovaries with cryoprotectant agent (CPA), rapid cooling, storage, thawing and, finally, dilution and removal of the CPA with return to physiological environment. Our study compared ISO cryo ovaries with cryo-diffused ovaries. We systematically examined the effects of ISO cryo and diffuse cryo on ovaries by morphological examination and with viability tests. The percentages of morphologically normal and viable follicular oocytes from ISO cryo were significantly higher than those that resulted from the cryo-diffused method (p<0.01). The quality of follicular oocytes from ISO cryo ovaries appeared better than that achieved from cryo-diffused ovaries. In conclusion, this study shows that ISO cryo is highly efficient for cryopreservation of oocytes and ovarian tissue