Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Cleavage Rate of in vitro Matured Oocytes Fertilized with Conventional Semen in Buffaloes

M.H. Pitroda2,*, K.P. Khillare1, M.B. Amle1, M.D. Meshram1, A.B. Mali1, M.N. Rangnekar1, S. Zawar1
1Krantisinh Nana Patil College of Veterinary Science, Shirwal, Satara, Maharashtra and Dr. Vijaypat Singhania, Centre of Excelle nce, Vadgaon, Rasai, Taluka- Shirur-412 211, Maharashtra, India.
21-B Miniland, Tankroad, Bhandup West, Mumbai-400 078, Maharashtra, India.
Background: In vitro embryo production in buffaloes has gained much importance in this current scenario due to ever increasing population and high demand of milk and meat. Slaughter house derived bubaline ovaries are a cheap and abundant source of cumulus oocyte complexes.

Methods: Oocytes from the buffalo ovarian follicles were recovered by aspiration technique as it facilitates quick recovery. Total 155 ovaries were used in the present study. Surface follicles were measured using vernier calliper and categorized into three groups viz. < 3 mm, 3-5 mm and > 5 mm based on follicular diameter and oocytes were processed for IVM, IVF and IVC using conventional non sorted semen.

Result: Overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94%, 8.14±0.60%, 5.35 ± 0.76%, respectively. Overall recovery rate of COCs was 38%. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26% (C), 18.33% (D) and 29.87% (E) respectively. Maturation rate of oocytes were 81.96 ± 2.70%. Fertilization rate was 74.98 ± 3.87%, Cleavage rate % was 40.84±2.51% and Blastocyst percentage was 21.57±1.75% respectively. Application of in vitro embryo production technique using slaughter house ovaries can salvage the genetic potential of bubaline species.
There are about 207 million buffalo population in the world out of which roughly 97% are found in the Asian region (Fao, 2020). Countries with largest number of dairy buffaloes are India, Pakistan, China, Egypt followed by Nepal (Fao, 2020). The total buffalo population in India is 109.85 million (Livestock Census, 2019) ranking number one in the world with huge genetic diversity and showing an increase of about 1% over previous (Livestock Census, 2012) where the population was 108.70 million.

There is an imminent need to utilize recent advances in the field of reproduction to improve the outcome of bubaline industry. Laboratory production of embryos allows mass scale production of embryos for practical and scientific purposes as well as for the exploitation of oocytes from donors. Buffalo females have few primordial viz. 12000 – 19000 follicles in cortical region of ovaries and have higher rate of follicular atresia (Jamil et al., 2008). Owing to the demand of technical expertise, cost involved in the oocyte recovery from live animals is quite high compared to slaughterhouse derived oocytes which are cheaper and easily available source of COCs for in vitro production of embryos.

Fertilization process is considered as the most critical step for IVEP procedure in buffalo, as cleavage rates are lower in this species (Neglia, 2003). Furthermore, blastocyst rate of buffalo is poor (20%) when compared to cattle (35-48%) (Liang et al., 2008). These factors limits the applicability of IVEP in bubaline species (Sadeesh, 2015). It is therefore need to improve IVEP technology in buffaloes for improvement of genetic potential of ND breeds. For production of embryos in vitro, combinations of techniques are required viz. collection of immature oocytes, In vitro Maturation, In vitro fertilization and subsequent In vitro Culture.

In vitro maturation is a method in which immature oocytes of dead animals are retrieved from the ovary using various methods such as aspiration, scoring, slicing, puncture or a combination of aspiration plus slicing method (Khan et al., 1997, Mehmood et al., 2011, Hammad et al., 2014) and are allowed to mature in the laboratory which is a crutial step for fertilization and subsequent embryonic development. Maturation rate in cattle and buffaloes is almost similar (94 v/s 87% respectively) but a slightly lower cleavage rate is observed in buffalo as compared to cattle (84 v/s 65%) (Gasparrini, 2002, Neglia, 2003).

In vitro fertilization has drawn interest of many researchers as it can salvage the genetic potential from infertile female and can also yield large number of embryos from slaughter house derived oocytes which may go waste otherwise (Kumar 2012). Also, slaughter house ovaries are the potential source of immature oocytes. Therefore, large number of oocytes can be easily obtained for standardizing and optimizing various reproductive biotechnological techniques.

The present study was conducted from the month of January 2020 to December 2020 by using 155 slaughter house derived ovaries for experiment at Department of Animal Reproduction, Gynaecology and Obstetrics, Krantisinh Nana Patil College of Veterinary Science, Shirwal, Dist. Satara; Maharashtra, India and Dr. Vijaypat Singhania, Centre of Excellence, Vadgaon, Rasai, Taluka- Shirur, Maharashtra, India.
IVM media, IVF media, IVC media, Mineral oil, Washmedia, Percoll, Aliquot H, Aliquot PHE, Sperm(Dilution for percoll), Water for Humidification, these chemicals and media were obtained from Vitrogen (Av. Cel. José Nogueira Terra, 203 Cravinhos - SP, Brasil), MOFA Oocyte recovery Media (Global, Verona, WI, USA) and plasticware were procured from ThermoFisher SCIENTIFIC(Mumbai, India), SEWA MEDICALS LTD.(Pune, India) and CORNING® (NY, USA).
Collection of ovaries
A total 155 ovaries were procured from the local slaughter house. Immediately after collection the ovaries were placed in a stainless steel thermos flask containing 0.9% normal saline solution along with Inj. Gentamycin @ 50µg/ml and were transported to laboratory within 2 hours of slaughter. Oocytes were collected by aspiration of follicles using 18 G needle attached to a 5 ml disposable syringe. The oocytes were searched and washed in MOFA oocyte recovery media. Grading was done after searching of oocytes. Oocyte evaluation and graded oocytes were examined under stereomicroscopy and classified based on their cumulus investment, compaction and ooplasm homogeneity according to (de Loos et al., 1992) as A, B, C, D and E respectively.
In vitro maturation of buffalo oocytes using vitrogen media (Alvarez-gallardo et al.,  2020).
Aspiration of oocyte and transferring COCs to MOFA oocytes recovery media and later in a centrifuge tube. Pour the contents of centrifuge tube into searching dish.Then search the oocytes from searching dish, put and wash them into 3 droplets of 70 μl of Oocyte washing medium (Vitrogen) and a drop of 100ul maturation mediain 60mm petri dish. Then finally put into maturation well and keep into Benchtop BT37 incubator at 5/6/90 gas (5% CO2 :6% O2: 90% N2 in Air) 38.5oC humidified atmosphere for 22-24 hrs.Within 20 minutes of aspiration place graded oocytes for IVM.

In vitro fertilization of buffalo oocytes using vitrogen media
Make IVF medium one day before. Make 3 droplets of 100μl of IVF media (Vitrogen) into 60 x 15 mm Petridishes for oocytes washing. Add 70μl of IVF medium + BO-Oil (~750μl) i.e. (35μl IVF media layered with 750 μl oil and then immerse the pipette tip and touch the 35μl drop and release 2nd 35μl droplet in order to increase the surface area for sperms to each well of Nunc 4 well plate (pipette 1ml sterile H2O incenter well for humidification purpose) and equilibrate in bench top BT37 incubator having trigas (5% CO2:6% O2: 90% N2 in air) 38.5oC humidified atmosphere one day before of fertilization.
Semen setup
Thaw the semen straw in thaw unit water bath at 37oC for 30 seconds and check sperm motility. The volume of sperm for fertilization i.e. 2x106 sperms add to IVF dish containing oocytes. After maturation, on 22th hr start the washing of matured oocytes into 3 droplets of 100 ul of IVF media, wash well then remove and place into final 70 ìl fertilization micro drop along with sperms in bench top BT37 incubator having trigas (5% CO2 : 6% O2 : 90% N2 in Air) at 38.5oC. Humidified atmosphere for 18-20 hrs.
In vitro culture of buffalo embryos using vitrogen media
At 20th hr post IVF, remove fertilized potential zygotes and wash through 4 drops of 70 μl of warmed drop of IVC medium. In 1st drop, wash the potential zygotes then remove the cumulus in 2nd drop with help 5ul pipette (remaining hard cumulus of potential zygotes remove with help of stripper having pipettetip diameter of 135 µm). Wash denuded potential zygotes through remaining 2 drops of pre warmed IVC media. After washing, culture the potential zygotes in designated Nunc well containing 500 μl IVC medium + 400 ul oil in Bench top BT37 incubator having trigas (5% CO2 : 6 % O2 : 90% N2 in air) at 38.5oC humidified atmosphere for 7 day until the embryo development.

The grading of follicles based on the dimensions of buffalo ovarian follicles in group I are depicted in Table 1. In group I where oocytes were subjected to fertilize with conventional non sorted semen, out of 155 ovaries 228 small follicles, 114 medium follicles and 75 large follicles were observed. The overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94, 8.14 ± 0.60, 5.35 ± 0.76, respectively as depicted in Table 1. A total, 417 surface follicles were measured using Vernier caliper out of 155 ovaries by categorizing them based on follicular size diameter as small, medium and large respectively.

Table 1: Group 1(n=155): Grading of buffalo ovarian follicle oocytes subjected to fertilize with conventional Non-sorted semen.

In the present study, the number of small, medium and large follicles were far more than the findings of Bhajoni et al., (2018) who reported 4.36 ± 0.31 small, 5.79 ± 0.75 medium and 0.82 ± 0.07 large follicles whereas Elbaz et al., (2019) reported 0.95 small, 0.57 medium and 0.24 large follicles respectively when follicles of 155 ovaries were measured using vernier calliper. This could be because of the negative effect of CL which was surpassed by the presence of good number of follicles on ovary in present trial.       
Aspiration of buffalo follicular oocytes and recovery percentage
The technique used in present study was aspiration of oocytes from the buffalo ovaries obtained from abattoir because it was more practical method than others which enabled quick collection of significant quantity of oocytes within no time (Katska., 1984, Totey et al., 1992, Mehmood et al., 2011, Rao and Uma Mahesh 2012, Shahid et al., 2014). No significant difference was observed in oocyte maturation, fertilization and development in vitro among the above mentioned methods of recovering oocytes (Pawshe et al., 1994). Thus in present study, comparatively practical method was used for recovery of COCs from ovaries.

Number of COCs recovered when oocytes were fertilized using conventional non sorted semen was 409 and mean oocytes recovered per trail were 29.21±1.58. Overall recovery rate of COCs (cumulus oocytes complexes) in conventional non sorted semen in vitro fertilization trail was 38% and mean oocytes recovery rate per trial was 2.74 ± 0.19% as depicted in Table 2.

Table 2: Group I: Total number of COCs recovered by aspiration of buffalo ovarian follicles and recovery rate% when oocytes were subjected to fertilize using conventional non sorted semen.

Khan et al. (1997) recovered 635 COCs from 192 ovaries; Nandi et al., (2000) recovered 425 COCs from 457 ovaries; Jamil et al., (2008) recovered 363 COCs from 298 ovaries of riverine buffaloes; Hammad et al., (2014) recovered 665 COCs from 206 ovaries of Egyptian buffaloes; and Elbaz et al., (2019) recovered 335 COCs from 187 ovaries.

In the present study, mean oocyte recovery rate per trail was 2.74±0.19%. These values were in accordance with Jamil et al., (2008) who reported oocytes recovery rate of 2.45±0.91% during peak breeding season and 1.93 ± 0.68% during low breeding season respectively.

Furthermore, Nandi et al., (2000) recovered lower oocytes 0.12% whereas Rao and Uma Mahesh, 2012 recorded higher oocytes 4.60 ± 0.33% than the present study.

Usually buffaloes are slaughtered because of unproductiveness and poor fertility (Selvaraj et al., 2008). The lower oocyte recovery rate in buffaloes may be due to anestrous condition as well as acyclicity and also because of presence of CL (Totey et al., 1992, Das et al., 1996). This is because follicular development is restricted when luteal cells occupy major part of the ovary (Kumar et al., 1997). Ovaries which do not possess CL have higher number of follicles being recovered and also higher grade COCs than those non CL bearing ovaries (Khandoker et al., 2011). Furthermore, the stroma of buffalo ovaries are thick and follicles are deeply embedded in them and therefore aspiration of oocytes from follicles gets difficult (Selvaraj et al., 2008, Hammad et al., 2014). The ovaries which had CL yielded lower number of oocytes as compared to non CL bearing ovary as it was discussed by (Kumar et al., 1997). The lower number could be because of greater mechanical damage induced on the granulose cells during the procedure of recovery.
Evaluation of oocytes and subsequent grading of COCs
Oocytes were examined under stereomicroscopy and classified according to their compaction, number of cumulus cell layers and homogeneity of ooplasm (Ravindranatha et al., 2003) into 5 categories as:
A)    Dense and compact multilayered with (≥5) cumulus layers with homogenous ooplasm.
B)    Dense and compact multilayered with (3-4) cumulus layers with homogenous ooplasm.
C)    Less compact (1-2) cumulus layers with homogenous ooplasm.
D)    Denuded cumulus layer with homogenous ooplasm.
E)    Denuded cumulus layer with unevenly granulated ooplasm.

Accordance to above criteria the oocytes were classified as A, B, C, D and E (De loos et al., 1992, Khan et al., 1997, Jamil et al., 2008).

In the present study, in conventional method, grade A oocytes were 5.07 ± 0.51, Grade B were 4.57 ± 0.56, Grade C were 7.21 ± 0.55, Grade D were 5.57 ± 0.66 and Grade E were 7.36 ± 0.83 respectively as depicted in Table 3. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26 % (C), 18.33% (D) and 29.87% (E) respectively.  

Table 3: Grading of Ovarian Oocytes Based on the Cumulus Layers Surrounding the Oocytes Subjected to Fertilize with Conventional Non Sorted Semen.

The oocyte quality plays major role in assessment of oocyte development therefore it is essential to grade the oocytes which in this study were correlated with (Elbaz et al., 2019) who got similar results, grade A oocytes were 5 ± 0.68, Grade B were 6.1 ± 1.1, Grade C were 4.8 ± 0.84 and Grade D were 4.8 ± 0.55 respectively.

Percentage of graded oocytes based on the compactness of cumulus layers in both the groups were in accordance with Khan et al., (1997) who recorded A (18.42%), B (15.43%), C (19.37%) and D (46.77%) whereas Samad et al., (1998) recoded A (18.42%), B (15.43%), C (19.57%) and D (46.77%) respectively. Bhajoni et al., (2018) recorded A (47.58%), B (37.42%), C (8.82%) and D (6.12%) oocytes which were higher than the present study and Nandi et al., (2000) recorded A (0.10%), B (0.13%) and C (0.11%) oocytes which were lower than the present study.

Maturation Rate of COC’s Subjected for Maturation using Conventional Non Sorted Semen
In the present study, average number of COCs used for IVM in 14 trials of non sorted group was 29.21±1.58. Maturation rate of oocytes were81.96 ± 2.70% in conventional non sorted trail as depicted in table 4. This maturation rate was in accordance with (Arul et al., 2017, Totey et al., 1992) who got maturation rate as 81.71 ± 1.81% and 81.7 ± 14.5% respectively as depicted in Plate 1.

Table 4: Maturation rate of COC’s subjected for maturation using conventional non sorted semen.

Plate 1: Cumulus expansion of oocytes post IVM in conventional non sorted semen trial.

Maturation rates obtained by (Kumar and Maurya 2000, Adlak et al., 2007, Leal et al., 2007 and Elbaz et al., 2019) was 53.35%, 71.05%, 36.68% and 28.82% respectively were lower than the present study. Furthermore maturation rates observed were higher in studies of (De matos et al., 1995, Umadevi et al., 1998 and Habeeb et al., 2018) was 90 ± 2 %, 83.33% and 86.5% respectively. This maturation rate was in accordance with (Suresh et al., 2009) who got maturation rate as 78.36 ± 2.16% respectively.

In conclusion, such variations in the maturation rates could be due to variations in the number of COCs used for IVM, variation in the choice of media used must have influenced maturation rates.

The average number of oocytes used for fertilization in conventional trial was 23.78±1.33 and fertilization rate was 74.98 ± 3.87% as depicted in Table 5.

Table 5: Fertilization rate of COC’s subjected for fertilization using conventional. non sorted semen.

Fertilization rates were in accordance with (Nain et al., 2006, Barkawi et al., 2007) who recorded 60.70% and 80.2% of fertilization rates. Furthermore, Bavister et al., 1992 recoded higher fertilization rates as 85±5% and (Jamil et al., 2008, Palta and Chauhan 1998 and Baczkowski et al., 2004) recorded 63.75 ± 2.81%, 40-55% and 57.1% respectively which were lower than present study.
Cleavage rate of potential zygotes in conventional unsorted group
In the present study, average number of potential zygotes used in IVC are 17.92 ±1.43 and the number of average cleaved embryos obtained were 7.35 ± 0.76 in conventional trial whereas cleavage rate % was 40.84±2.51% as depicted in Table 6 which was in accordance with Chauhan et al., (1998) and Nandi et al., (2000) who observed cleavage rate of 40%

Table 6: Number of COC’s subjected for IVC and their subsequent cleavage rate in conventional non sorted semen trial.

Cleavage rate was in accordance with (Nain et al., 2006 and Aquino et al., 2013) was 40-50% and 40% respectively. Cleavage rates observed (Madan et al., 1994, Mehmood et al., 2007, Suresh et al., 2009 and Gonclaves et al., 2013) were higher 54.4%, 75.9%, 52.14 ± 1.15% and 71.8% respectively. Furthermore, cleavage rates observed (Ocampo et al., 2015, Sadeesh et al., 2016) were lower 39.6%, 22.5±4.2% respectively than present study as depicted in Plate 2.

Plate 2: Cleavage of potential zygotes observed on day 4 post in vitro fertilization in conventional non sorted semen trial.

In the present study, average number of blastocyst formed were 3.92±0.46 and blastocyst percentage was 21.57±1.75% as depicted in Table 7 which was in accordance with (Neglia et al., 2003) who observed blastocyst % of 19.9 ± 4.2%. Blastocyst % observed was in accordance with (Ocampo et al., 2016 and Sadeesh et al., 2016) as 19.6%, 20.4±2.5% respectively. Blastocyst percentage was higher than present study (Aquino et al., 2013, Ocampo et al., 2015 and Shabankareh et al., 2015) as 32.50%, 22.5% and 38.7% respectively and lower than present study (Palta and Chauhan et al., 1998, Boni et al., 1999, Prasad et al., 2013) as 10-15%, 13.5%, 10.65 ± 0.45% respectively in Plate 3.

Table 7: Number of blastocyst formed and blastocyst percentage of embryos in conventional unsorted group.

Plate 3: Blastocyst formed on day 7 post in vitro culture in conventional non sorted semen trial.

In vitro fertilization of oocytes recovered from slaughter house ovaries is the cheapest method of production of large number of embryos using conventional semen for in vitro embryo production in buffaloes.

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