Dairy cow is essential for reducing rural poverty and it is the backbone of Indian dairy industry. India is the world’s highest milk producer country contributing over 25% of the world’s milk production. One of the important objectives was Conservation and multiplication of superior germplasm in Indigenous cattle by OPU-IVF technology and standardization of IVF technique (Deshpande et al., 2023). Comparatively, Indigenous breeds possess superior thermo-tolerance and the ability to maintain normal biological functions under adverse environmental conditions than exotic breeds (Srivastava et al., 2019). In vitro fertilization (IVF) with Embryo Transfer (ET) technology has been introduced to conserve the cow breeds by increasing their reproductive efficiency (Nandi et al., 2002), enhance the milk production and simultaneously ensure rapid multiplication of superior germplasm. Sire contributes to 50% of the nuclear genetic makeup but essentially 0% of the mitochondrial genetic makeup in an offspring. Sire’s nuclear genome must be compatible with the dam’s inherited mtDNA for optimal metabolic health. The successful outcome of IVF was depended mainly on gametes quality. Sperm separation methods improve the sperm quality with higher rate of progressive motility and morphologically normal spermatozoa. Such selection of spermatozoa separates motile sperm from nonmotile, removes seminal plasma, cryo-protective and infectious agents, other background materials and debris (Henkel et al., 2003) and initiates the capacitation of sperm (Centola et al., 1998).
       
The less selective environment of in vitro production compared to the female reproductive tract allows for the survival and development of lower-quality bovine embryos that would typically be eliminated by natural maternal filters. This absence of rigorous biological control in the oviduct and uterus often leads to embryos with reduced viability, altered gene expression and lower pregnancy rates (Van Soom et al., 2007). Both fertilization failure and failure in subsequent in vitro bovine embryonic development are of seminal origin (Saacke et al., 2000). The in vitro blastocyst and pregnancy rates remain considerably lower than those obtained for embryos produced in vivo (Hansen et al., 2006). The present study was designed to compare the efficiency of sperm separation methods i.e., Density gradient (Percoll) and Swim-up methods in terms of evaluating sperm microscopic parameters i.e., motility, viability, plasma membrane integrity and DNA integrity and subsequent developmental rate of in vitro embryos.

The present study was carried out at the in vitro Fertilization-Embryo Transfer Technology Laboratory, Department of Animal Reproduction, Gynaecology and Obstetrics, Nagpur Veterinary College, Nagpur from October 2024 to January 2025.
       
A total of 10 high-yielder Sahiwal cows with annual lactation yield of >3500 litre, BCS of  more than 3 (scale 1-5) and free from any infectious diseases were selected as Donor cows. Ovum pick-up (OPU) was performed in FSH stimulated donor cows with the help of transvaginal ultrasound probe having oocyte aspiration assembly along with aspiration pump (WTA, Brazil). Following epidural anaesthesia, the transducer (Exago, IMV, France) was inserted into the vaginal canal and fixed in the vaginal fornix and the ovary was positioned against the head of the transducer per rectally. All visible follicles were aspirated by using a portable scanner that had a 5.0 MHz convex transvaginal array transducer and a stainless-steel needle guide (18 G; WTA, Brazil).The aspiration needle was attached to aspiration equipment and a vacuum system at negative pressure of 70 mm Hg. A tubing system of 1.1 mm inner diameter, 120 cm long (WTA, Brazil) was connected to a 50 mL conical tube containing 15 mL of OPU media kept at 37°C.
       
After filtration through 75 µm filter (WTA, Brazil) and Euroflush media (IMV, France), the retrieved follicular content was examined under stereo zoom microscope. The collected oocytes of Grades A, B and C in the petri dish were subjected to in vitro maturation for next 22-24 hours in benchtop incubator. The matured oocytes were washed and transferred to IVF medium (Vitrogen, Brazil). The concentration of spermatozoa in frozen straws was 20 million/ mL with initial motility (>70-80%). The semen straw of pedigree bull was thawed. The Density gradient and Swim-up semen separation methods were carried out as followed.
 
Density gradient method (Percoll method)
 
This method has been commonly used to clean out dead sperms in reproductive laboratory using only 2 different density layers of solution (Promthep et al., 2016). Semen undergone specific preparation steps-primarily thawing and often washing or dilution before being layered onto the density gradient.  
 
Preparation of 10× concentrated salt solution (Buffer)
 
• Composition: 2.889 g NaCl, 0.238 g KCl, 0.116 g KH₂PO₄, 0.112 g CaCl‚  and 0.163 g HEPES Buffer dissolved in 50 mL distilled water.
• Alternatively, 10× Sperm-TALP or PBS (consisting of 1.37 M NaCl and 27 mM KCl) can be used. The 10× solution was filtered and autoclaved (121°C for 20 min) and stored  at 4°C. 
       
To make the 100% Isotonic Percoll (100% SIP), mix Percoll with the 10× buffer at a 9:1 ratio. The final density should be ~1.123 g/mL (in saline) and osmolality was adjusted to 280-320 mOsm/kg.
       
The 100% SIP is diluted using a standard sperm washing medium, such as Hepes-TALP or BO medium. 
• 90% Percoll Solution (Bottom Layer): Mix 9 parts of 100% Isotonic Percoll (SIP) with 1 part of washing medium (e.g., 9 mL SIP + 1 mL Hepes-TALP).
• 45% Percoll Solution (Top Layer): Mix 1 part of the 90% Percoll solution with 1 part of washing medium (e.g., 1 mL 90% Percoll + 1 mL Hepes-TALP).
       
Frozen thawed semen was layered on the gradient prepared using upper and lower isolates of percoll in a 2.00 mL microcentrifuge tube and the mixture was centrifuged at 4700 rpm for 6 minutes. After discarding the supernatant, the pellet was aspirated and transferred to the pre incubated IVF medium and centrifuged again at 1700 rpm for 3 minutes. The sperm pellet was mixed thoroughly and the washed sperms were inoculated in the IVF droplets containing the matured oocytes.  Density gradient centrifugation was also used for sexing spermatozoa because of the additional DNA content and volume of X-bearing sperm head (Bhat et al., 2020). 
 
Swim-up method
 
2 ml of diluted semen was taken in Falcon tube and centrifuged at 500 G for 5 minutes. The sperm pellet was resuspended in 1 mL TCM-199 with antibiotics which was then placed at the bottom of four falcon tubes (0.25 mL each) and 1 mL of TCM-199 medium was layered in each Falcon tube above the suspension. Each tube was then incubated at 38.5°C for 1 hour for swim-up. Further, the top 0.25 mL layer from each of the four tubes was removed and pooled together in one tube. The layer resuspended in 1 mL of TCM-199. To this 10 µg/mL heparin was added and placed in incubator. After semen separation by swim-up method, the petri plate containing the oocytes and sperm was incubated in a benchtop incubator at 38.5°C.
       
The microscopic semen parameters like Motility, Viability by Eosin-Nigrosin staining (Fig 1), Plasma membrane integrity by Hypo-osmotic swelling test (Fig 2) and DNA fragmentation index by acridine orange test (Fig 3) were studied post thaw and with the pellet obtained after density gradient method and swim up method. After 18 hours, the presumptive zygotes were washed and the connected cumulus cells were removed using a 140 µm diameter glass pipette attached to stripper (Cook, Australia). The presumptive zygotes were transferred to the IVC medium (Vitrogen, Brazil) for the next seven days.






       
On 7^th day of IVC, the blastocyst production rate following IVF with semen separated by density gradient and swim-up method was assessed accordingly. The embryo transfer was done in close to the tip of the ipsilateral horn to the side of corpus luteum in recipient cows. Pregnancy diagnosis was carried out transrectally using ultrasound machine (Z5 Vet, Mindray, China) at 45 days of embryo transfer in the recipients. Conception rate following transfer of blastocysts in both the treatment groups was compared accordingly. The collected data was statistically assessed per the WASP 2.00 ICAR, Goa.

As shown in Table 1, ten ovum pick up sessions were conducted each in Group-I and Group-II methods aspirating total 328 oocytes. These oocytes were in vitro matured and in vitro fertilized using the semen sample prepared by Group-I and Group-II methods. Subsequently, different microscopic characteristics of frozen thawed semen were studied.


       
The present findings of semen motility following separation by density gradient method and swim-up method are in accordance with the earlier findings of García-López et al. (1996) in ram, Palomo et al. (1999) in bull, Rho et al., (2001), Trentalance et al., (2002) and Jamil et al. (2007) who stated that percoll gradient method is the best method for separation of highly motile spermatozoa from the poor-quality semen post thaw. Viability of semen showed significantly higher sperm viability following semen separation by density gradient method than swim-up method. Similar findings are reported by Valcarcel et al. (1996); Somfai et al., (2002); Trentalance et al., (2002) and Jamil et al. (2007). Significantly higher plasma membrane intact spermatozoa is observed following semen separation by density gradient method than the swim-up method. Valcarcel et al. (1996), Samardzija et al., (2006) and Jamil et al., (2007) also recorded the similar findings and hence are in agreement with the findings of present study.
       
On day 7^th of the in vitro culture, the overall blastocysts conversion rate of 98 (60.87%) and 46 (27.54%) was noted in Group-I and Group-II methods. In the present study, significantly higher blastocysts conversion rate was recorded in density gradient method (Group-I) than in swim up method of semen separation. Seidel et al. (1995) and Larocca et al. (1997) also recorded that density gradient method yields better blastocysts yield than swim-up method. Rho et al. (1998); Rho et al. (2001) and Matás et al. (2003) noted that density gradient method separated the highly motile population of spermatozoa, thus yields higher blastocysts conversion rate as compared to other methods of semen separation.
       
Following transfer of in vitro fertilized embryos (Fig 4) by the semen sample prepared by Density gradient (Group - I) and Swim-up method (Group-II) of semen separation, average conception rate of 27.55% (27 pregnancies) and 15.22% (7 pregnancies) had been reported in the present study. Larocca et al. (1997);  Alomar et al., (2006) and Cesari et al., (2006) also reported improved conception rate following use of semen separated by density gradient method which is in accordance with the findings of present study.

Following semen separation by Density gradient method (Group - I), significantly higher semen motility, viability, plasma membrane integrity and lower DNA fragmentation index was observed than in semen separation by Swim-up method (Group - II). Also, significantly higher blastocyst yield  and conception rate had been reported with Density gradient (Group - I) than in Swim-up (Group - II) method of semen separation.

The present study was supported by all the respective authors.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily represent the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided, but do not accept any liability for any direct or indirect losses resulting from the use of this content.
 
Informed consent
 
All animal procedures for experiments were approved by the Committee of Experimental Animal care and handling Techniques were approved by the University of Animal Care Committee.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.