Comparison of culture media for their effects on development of caprine IVF embryos using fresh and cryopreserved semen

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1 Indian J. Anim. Res., 50 (6) 2016 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Comparison of culture media for their effects on development of caprine IVF embryos using fresh and cryopreserved semen Ajay Pratap Singh, Dharmendra Kumar, Gopalakrishna R, Rakesh Ranjan, Saurabh K Pandey and Bikash C Sarkhel* Animal Biotechnology Centre, Nanaji Deshmukh Veterinary Science University, Jabalpur , India. Received: Accepted: DOI: /ijar.5709 ABSTRACT The aim of the present study was to compare the effects of two culture media on in vitro fertilized embryo produced by fresh and cryopreserved buck semen respectively. The in vitro fertilized embryos from both semen sources were cultured separately in the two media groups viz., Research Vitro Cleave-BSA (RVCL-BSA) and RVCL-Blast-BSA media. Under RVCL BSA medium, the fresh semen produced higher rates of cleavage (68.94±1.86 vs 65.62±2.34) and significantly higher (P 0.05) morula (27.89±1.18 vs 23.84±2.23), blastocysts (15.26±0.67 vs 13.07±1.17) and blastomeric counts (232.65±14.26 vs ± 10.26) than cryopreserved semen. The similar developmental patterns were exhibited in RVCL-Blast-BSA medium in which the fresh semen showed significantly higher (P 0.05) rates of morula (38.94 ± 2.37 vs 31.87±2.10), blastocysts (29.89 ± 2.02 vs ± 1.78) and blastomeric count ( ± vs ± 14.38). This study concludes that the RVCL-Blast-BSA medium produced significantly higher (P 0.05) embryonic developmental rates in comparison to RVCL BSA medium irrespective of semen sources and the fresh semen afforded better fertilization rates than cryopreserved semen in both culture groups. Key words: Blastocyst, Cryopreservation, Embryo culture, In vitro fertilization, Semen. INTRODUCTION Assisted reproductive technologies (ART) are considered a major tool for accelerating the genetic improvements in many animal species. ART includes in vitro fertilization (IVF), intra cytoplasmic sperm injection (ICSI), somatic cell nuclear transfer (SCNT) etc. In vitro techniques for oocytes maturation and fertilization are receiving more attention in small ruminant species such as in the sheep and goat than in large species due to their short generation intervals, prolificacy and economical rearing (Kharche et al. 2009). Although recent efforts in caprine, in vitro embryo production have led to advances in many steps, the efficiency of in vitro production is still low with regards to obtaining fully matured oocytes and culture of embryos in vitro in terms of blastocyst formation. It is most likely due to the fact that the in vitro environment cannot mimic in vivo and thus results in inefficient embryo production and their subsequent developmental potential (Fleming et al. 2004). The improvement of the caprine culture system is highly desirable in terms of the production of pre-implantation stage embryos for both biotechnological studies and embryo transfer techniques. In vitro fertilization involves the union of capacitated sperm with matured oocytes to form a zygote. As an alternative to fresh semen, cryopreserved semen is also being used within IVF experiments in both livestock and human *Corresponding author s sarkhelbc@yahoo.co.in settings. Cryopreservation is a potentially useful way of banking sperm until needed for experimentation. Long term preservation of spermatozoa in liquid nitrogen is a subject of paramount interest because of the extensive use of frozen semen for IVF in livestock breeding. Although different cryopreservation methods are useful, some limitations exist because of the lower recovery of motile spermatozoa and fertilization efficiency as compared to fresh semen (Romao et al. 2013). In most mammalian species, the recovery rate in terms of motility, ranges between 50-60% (Dorado et al. 2010, Bispo et al., 2011). During cryopreservation a substantial portion of sperm undergo damage from thermal, mechanical, chemical and osmotic stresses (Rodojcic et al. 1998; Colas et al. 2009; Watson et al. 2000). In the last few years, the considerable increase in our understanding both of the cell physiology of spermatozoa and of the stresses of cryopreservation has contributed to a renewed interest in improving the performance of cryopreserved semen. In view of the above, the present work was designed to optimize culture medium for caprine embryo production by IVF using fresh and cryopreserved semen and to utilize embryos for genetic improvements of goat breeds.. Therefore a detailed study was conducted to assess the effects of two different culture media for their respective impacts to the production of caprine IVF embryos utilizing both fresh and cryopreserved semen.

2 MATERIALS AND METHODS All plastic wares were purchased from Falconware Becton-Dickinson (Bedford, MA, USA) and chemicals/ biochemicals from Sigma Chemical Co. (St. Louis, MO, USA), unless otherwise specified. Oocytes recovery and in vitro maturation: Goat ovaries were collected from the abattoir and transported to the research station in 0.9% normal saline at 38.5 C within 3-4 h. The experiment was performed for the period of five months i.e. February to June including three months of breeding and two months of non-breeding season of goat. The ovaries were properly trimmed and washed 5 times in sterile Dulbecco s phosphate buffer saline (DPBS). Cumulus oocytes complexes (COC s) were aspirated from 4-8 mm sized follicles. The COC s having 3 cumulus layers and homogenous ooplasm were allowed to mature in groups of numbers into 50 µl drops of maturation medium (TCM- 199, Hyclone) fortified with 7.5% (v/v) fetal bovine serum (Hyclone), 2.5 unit/ml follicle stimulating hormone, 2.5 unit/ ml leuteinizing hormone, 1 µg/ml estradiol, 0.8 mm/ml sodium pyruvate and 50 µg/ml gentamicin in 5% CO 2 at 38.5 C for 27 h. Semen collection and evaluation: In the present experiment, single superior buck was selected to avoid buck to buck variability in terms of semen production, freezabilty and its efficiency for production of IVF embryos. Fresh semen was collected from selected buck by artificial vagina method. The buck was used twice a week having a rest period of three days between collection. Immediately after collection, the semen volume, color, concentration, morphology, initial and gross motility of sperm were assessed. Cryopreservation of semen: The seminal plasma was immediately removed by diluting 1 ml semen with 9 ml of pre-equilibrated DPBS, followed by centrifugation at 1000 rpm for 8 min. The semen pellet was mixed with 5 ml of preequilibrated sodium citrate-egg yolk extender. The temperature of the suspension was gradually reduced from 38.5ºC to 4ºC in a water bath within a period of approximately 90 min and kept for 2 h. The semen was further diluted with an equal volume of pre-cooled sodium citrate-egg yolkglycerol (14 %) extender and kept for another 4 h at 4ºC. Through the open end of 0.25 ml straws, semen was filled with a sterile spinal needle (20 G x 89 mm) attached to a disposable syringe, leaving about 0.5 cm air space at open end. The open end was sealed using hot artery forcep. During the process of filling and sealing of straws, the temperature of straws and semen were strictly maintained at 4º C. For further cooling, an indigenously designed assembly was used which consisted of a clean and sterile thermocole container of 15 cm height and 20 cm diameter. The container was half filled with liquid nitrogen (LN 2 ). A wire-mesh rack compatible with the diameter of the container was placed at 5 cm above the level of LN 2. Filled straws were placed evenly and Volume 50 Issue 6 (2016) 847 horizontally over the wire-mesh rack to allow the exposure of LN 2 vapors for min or until a cracking sound heard. The rack was then gradually lowered down and ultimately plunged into the LN 2. The straws were then transferred into LN 2 container. After 24h, 2-3 straws were thawed to check the viability and motility of sperm. Sperm treatments and IVF: The fresh and frozen semen of the same buck was used for all IVF throughout within the experiment. In case of cryopreserved semen, the semen straw was thawed and sperm examined for morphology, viability and motility under an inverted microscope on a glass slide. To remove the cryoprotectants, the thawed semen was diluted with 10 ml Bracket and Oliphant (BO) medium and centrifuged at 1000 rpm for 8 min. The pellet was washed firstly with 10 ml BO medium supplemented with 0.3% bovine serum albumin (BSA) and then with 9 ml fertilization media (BO medium containing 0.6% BSA and 20 µg/ml heparin). Finally, the sperm pellet was diluted with 2 ml of fertilization medium and transferred into a micro centrifuge tube held at 45 angle at 38.5 C for 1 h. Simultaneously, the in vitro matured oocytes were transferred into 50 µl microdrops of fertilization medium. From the top layer of semen samples, aliquots of sperm suspension were added to the droplets to give a final concentration of 10 6 sperm/ml in each 50 µl droplet containing 10 matured oocytes. Oocytes were co-incubated with sperm at 38.5 C in 5 % CO 2 for 18 h. In case of fresh semen, the collection and routine examination of semen and removal of seminal plasma from fresh buck semen were carried out as per the procedure described earlier. The 1 ml sperm pellet was equally distributed into five micro centrifuge tubes. Inside each tube, 1 ml BO medium was overlaid and incubated at 38.5 C for 1 h. The top 0.7 ml of each tube was pooled into a sterile 15 ml centrifuged tube. The sperm washing and treatments were given as same procedure described for cryopreserved semen except, the heparin concentration in fertilization media was 50 µg/ml. Embryo culture: After 18 h of co-incubation, oocytes to be utilized with either semen sources (fresh or cryopreserved) were denuded separately with slight agitation by fine bored pasteur pipette and washed five times with pre-equilibrated Research Vitro Cleave media (RVCL, Cook, Australia) supplemented with 1% fatty acids free bovine serum albumin (BSA). Two types of culture media were used for embryo culture. In the first group, the presumptive embryos were cultured in RVCL medium with a 1 % BSA (RVCL-BSA) for 8 days in a CO 2 incubator having 5% CO 2 at 38.5ºC. In the second group, the embryos were cultured initially in RVCL- BSA medium for 3 d and further cultured in Blastocyst medium (Cook, Australia) with a 1 % BSA (RVCL-Blast- BSA) for 5 d under the same culture conditions. For each IVF experimental group, embryos were assessed for cleavage rate and subsequently for development to morula, blastocyst/

3 848 INDIAN JOURNAL OF ANIMAL RESEARCH hatched blastocyst stage produced by either fresh or cryopreserved semen. Embryo staining: All the blastocysts of different groups were washed twice in 50 l drops of PBS containing 1 mg/ ml polyvinylpyrolidone (PVP) and subsequently were transferred to a 50 l drop of Hoechst stain (10 µg/ml in PBS) for 10 min in a dark chamber. The blastocysts were kept on a glass slide with a coverslip for the blastomere count under fluorescence microscope examination. Statistical analysis: Six replicates of IVF experiment with each of the fresh and cryopreserved semen sources under two culture media were conducted. The effect of culture media on IVF embryos obtained by both semen sources were statistically analyzed by hierarchal ANOVA and the significant difference between mean values of each group was tested by Duncan s multiple range test and compared at 5% level of significance (p <0.05). RESULTS AND DISCUSSION In vitro maturation: In the present study, 2814 oocytes were aspirated from 994 goat ovaries and a total of 1435 (50.9%) oocytes exhibited good cumulus expansion were subsequently used for IVF. Embryo development: In RVCL-BSA culture group, the cleavage rates were higher in fresh semen (68.94±1.86) than cryopreserved semen (65.62±2.34). Similarly, significantly higher (P 0.05) morula (27.89±1.18 vs 23.84±2.23), higher blastocyst (15.26±0.67 vs 13.07±1.17) rate and significantly higher (P 0.05) average blastomeric count (232.65±14.26 vs ± 10.26) were found in fresh semen as compared to cryopreserved semen (Fig 1A, B, C, D, E, F, G, H). Similar patterns of developmental rates were observed under RVCL- Blast-BSA medium. In this group, fresh semen showed higher cleavage rates (69.05±2.12) than cryopreserved semen (65.62±2.34). The morula (38.94 ± 2.37 vs 31.87±2.10), blastocyst rate (29.89 ± 2.02 vs ± 1.78) and blastomeric count ( ± vs ± 14.38) were significantly higher in fresh semen as compared to cryopreserved semen (P 0.05). It is evident from table 1, that RVCL-Blast-BSA medium showed significant higher morula, blastocysts rate Table 1: Effect of culture media and semen sources on embryonic development of IVF caprine Culture media Semen source Oocytes Cleavage (%) Morula (%) Blastocyst (%) Blastomere counts inseminated RVCL-BSA Fresh 380 (68.94±1.86 a ) (27.89±1.18 a ) (15.26±0.67 a ) ±14.26 a Cryopreserved 260 (65.38±2.65 a ) (23.84±2.23 b ) (13.07±1.17 b ) ±10.26 b RVCL-Blast-BSA Fresh 475 (69.05±2.12 a ) (38.94±2.37 c ) (29.89±2.02 c ) ±16.54 c Cryopreserved 320 (65.62±2.34 a ) (31.87±2.10 d ) (25.31±1.78 c ) ±14.38b d Values with different superscripts within columns differ significantly (P 0.05) A X 200 C X 200 E X 200 G X 200 B X 200 D X 200 F X 200 H X 200 Figure Caption Fig 1: Figure showing different developmental stages of IVF embryos: (A)16-32 cell stage, (B) late morula, (C) Expanded blastocyst, (D) Group of advanced embryos showing expanded and hatching blastocysts, (E) One hatching blastocyst, (F) One hatched blastocyst, (G )& (H) Blastocyst showing Hoechst stained nuclei. and blastomeres count than RVCL-BSA, irrespective of semen sources (P 0.05). An indigenous and simple technique for cryopreservation of goat semen was used in this study. The viability of frozen-thawed spermatozoa by this protocol ranged within 50 to 60%. The capacitation of semen is a crucial process in which mammalian spermatozoa must undergo in order to achieve fertilizing ability. Heparin is the most commonly used chemicals as a capacitating agent for goat semen which stimulates the sperm capacitation, fertilization, cleavage and embryo developmental kinetics (Mendes et al. 2003). In the present IVF study, heparin was used at concentration of 20 µg/ml and 50 µg/ml for both frozen and fresh semen samples respectively. Several reports showing the use of varying concentration of heparin, ranging from 2 to 100 µg/ml, are documented in the literature (Cognie et al. 1995; Ongeri et al. 2001). In the present study, fresh semen showed higher embryonic developmental rates than frozen-thawed semen in both culture media groups. However, the reports on superiority of fresh or frozen-thawed spermatozoa for in vitro embryo production (IVEP) are debatable. Romao et al. (2013)

4 reported that fresh sperms improves the developmental competence and morphological qualities of IVF embryos whereas, Katska-Ksiazkiewicz et. al. (2004) reported that frozen-thawed sperms yields increased number of blastocysts. The storage of semen particularly in a frozen state, causes ultrastructural, biochemical and functional damage to the spermatozoa resulting in a reduction of motility, viability and fertility (Leboeuf et al. 2000, Medeiros et al. 2002). The lower fertility during IVF and subsequent embryo development using frozen semen might be due to presence of egg yolk in cryopreservation media (Leboeuf et al. 2000). Seminal plasma of caprine contains phospholipase A and triglycerol lipase which are secreted from bulbourethral gland. These enzymes catalyze the hydrolysis of egg yolk lecithin into fatty acid and lysolecithin (Pellicer, 1995). Lysolecithin being toxic to goat spermatozoa may be responsible for the decreased survivability and reduced quality of spermatozoa during cryopreservation. The successful production of advanced stages of embryos critically depends upon culture conditions and media formulations that have been optimized to mimic the in vivo environment (Summers and Biggers, 2003). In the present study, the IVF embryos produced by two sources of semen were cultured in commercially available media: RVCL and Blastocyst media. The results revealed that the developmental rate of morula and blastocyst rate, alongwith blastomere count, were significantly higher in the RVCL-Blast-BSA group than RVCL alone, irrespective of semen sources. The use of RVCL as embryo culture medium in livestock has been reported by numerous groups (Shah et al. 2008, Jena et al and Kumar et al. 2014). Shah et al. (2008) reported that RVCL medium showed improved results compared with CR2 and msof media in term of buffalo cloned embryo production. In the goat, Jena et al. (2012) has also reported a higher developmental rate of cloned and parthenogenetic embryos using RVCL medium as compared to msof and EDM (Embryo development medium). The comparative study of RVCL and RVCL- Blast media has been done Volume 50 Issue 6 (2016) 849 previously in our laboratory, which is the only published report available in literature (Kumar et al., 2014 and Gopalakrishna et al., 2014). It was highlighted that the combinations of RVCL and Blastocyst media enhanced the embryonic development by providing the embryotrophic effect in a stage specific manner. Thus, the development of caprine parthenogenetic embryos observed in RVCL-Blast media was substantially enhanced as compared to RVCL medium alone, which might be due to availability of additional beneficial components for supporting the in vitro development of embryos during later stages. The increased concentration of glucose found in the blastocyst medium (RVCL-Blast- BSA) is considered as one of the important factors responsible for supporting the embryonic development to blastocyst as evidenced by the stage specific requirement of energy molecules for embryonic development has been emphasized by Lane and Gardner (2007). In the case of bovine, it has been found that glucose inhibits early embryonic development whereas for later stages (day 3 onward) higher level of glucose are required (Chatot et al. 1989, Gordon 2003, Kharche et al. 2011). Apart from glucose, blastocyst medium is also fortified with essential and non essential amino acids and pyruvate that is optimized for embryonic development on later stages. CONCLUSION In the present study, two culture media were compared on the basis of developmental potency of IVF embryos derived from fresh and cryopreserved semen sources. The RVCL-Blast-BSA medium was found to be superior to RVCL-BSA medium in terms of cleavage rate, morula, blastocyst developmental rate and blastomeric counts irrespective of semen sources. Also, fresh semen proved to be a more efficient source for in vitro embryo production than cryopreserved semen. ACKNOWLEDGEMENT The authors are thankful for the financial support provided by Department of Biotechnology, Government of India, New Delhi as Junior Research Fellowship to the author. REFERENCES Bispo, C.A.S., G. Pugliesi., P. Galvao., M.T. Rodrigues., P.G. Ker., B. Filgueiras. and G.R. Carvalho. (2011). 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