Adaptation of In -vitro maturation, fertilization and embryo production, Oocyte/embryo cryopreservation and vitrification and Tubal Embryo Transfer techniques and its Impacts on reproductive and productive performance in Dairy Animals

Adaptation of In -vitro maturation, fertilization and embryo production, Oocyte/embryo cryopreservation and vitrification and Tubal Embryo Transfer techniques and its Impacts on reproductive and productive performance in Dairy Animals

The extensive investigations of mammalian embryo conducted during the last three decades have increased our depth of understanding of normal biophysiologic events that take place during fertilization and early embryonic development. Species difference have been marked in characteristic of estrous cycle, ovulatory mechanism, times of ovulation, viability of egg, time of cleavage, stage and location of morula and blastocyst in the female reproductive tract. Although, the mammalian ovaries contains hundred of thousand oocytes, the number of progeny a female produced is small. In farm animal the number of times a female can become pregnant   is severely limited by the extended duration of gestation. In-vitro techniques are powerful tools for studying physiology of maturation, fertilization, development of periimplantation embryos and increasing production as it give access to micromanipulation of embryos.

The technique of IVMFC essentially involve collection of oocytes from slaughter house ovaries or from living animals through transvaginal aspiration, in-vitro maturation of selected oocytes in media containing serum and hormones, in-vitro fertilization either with frozen thawed semen or freshly collected in-vitro capacitated spermatozoa and in-vitro culture of embryos in media containing oviduct or cumulus cell up to the morulae or blastocyst stage, at which stage they can be either transferred to synchronized  recipients or frozen and stored ( Palta and Chauhan , 1998 ).

The potential for commercial production of genetically superior embryos by in vitro ferti1ization is apparent. In the past few decades there have been unprecedented evolution of technology for in vitro embryo production of farm animals, with the rate of progress getting intensified in  the  last  decade  with  the  characterization  of effectively defined and semi defined medium for different species. The first succeeded IVF was achieved in rabbits in 1959 (Chang, 1959), next success was with mice in 1968. The first IVF production in human was in 1978 (Steptoe, 1980) and the first born calf produced with IVF was in 1981 (Brackett et al., 1982).  In vitro production technologies not only help  in  production  of high  genetic merit  animals  but  also  provide  an  excellent  source  of  embryos  for  emerging  biotechnologies  like  embryo  sexing, cloning,  nuclear  transfer, transgenesis etc. Furthermore, it allows analyzing developmental potential of embryos, including the pattern of gene expression, epigenetic modifications and cytogenetic disorders during the development  (Galli  and  Lazzari,  2008).  Early stages of  bovine embryo development  show  many  similarities  with  human  embryos.  Therefore,  bovine embryos are used as a model organism (Niemann and Wrenzycki, 2000). In spite of continuous  efforts to improve bovine in vitro embryo  production (IVP), its efficiency is still low, since only 30 to 40% blastocyst development has been obtained from oocytes after in vitro maturation, fertilization and embryo culture (Sirad et al., 2006). Despite of several advantages of IVP, initial application in both cattle and  buffaloes has  been  limited  by the  ability to  recover  oocytes.  However,  recent development of low invasive ultra sound guided transvaginal oocyte retrieval (TVOR) and oocyte pick up (OPU) has removed these difficulties to a large extent. Through OPU oocytes may be aspirated from live animals. Now OPU-IVP is used in many countries for the large scale embryo production at commercial  level. This  repeated recovery  permits production  of more  embryosthan  might be  possible by standard embryo  transfer  (ET)  practice  (Galli  and  Lazzari,  2008).  The  TVOR  also  allows repeated collection of oocytes from endangered species of livestock or livestock of high economic importance in order to propagate such genetic resources in much faster way. However, the practicaluse of IVEP is limited by high production costs and the low overall efficiency under field conditions.

 

Methods for in vitro maturation (IVM), fertilization (IVF) and culture (IVC)

 

Crucial steps of in vitro embryo production technique includes:

1. Oocyte collection (oocyte  pick up  from living  animal  or  post-motrem  from

slaughter houses).

2. Selecting and cleaning oocytes and placing oocytes in maturation medium for

18-24 h.

3. Sperm purification using percoll gradient.
4. Inseminate matured oocytes with purified sperm cells for 8-24h.
5. Removal of Cumulus Cells Complexes [can be done by mechanical (vortexing, pipeting) or by enzymatic digestion.
6. Placing putative zygotes in culture medium for 7-9 days.
7. Obtaining early bovine embryos that is ready to  be  transfer  to  surrogate  mothers.

Oocytes collection from Ovum Pick Up technique

The technique of ultrasound-guided transvaginal follicular aspiration for ovum pick-up (OPU), is a non-invasive procedure for recovering oocytes from antral follicles in live animals. In 1987, an ultrasonic-guided aspiration of bovine follicular oocytes was first proposed in Denmark and in 1988; a real OPU was first established in cattle by a Dutch. Together with in vitro fertilization of oocytes, OPU has been taken as a most flexible and repeatable technique to produce embryos from any given live donor. Unlike MOET, OPU does not interfere with the normal reproduction and production cycles of the donor. Any female starting from 6 months of age to the third month of pregnancy and even soon after calving (2-3 weeks) could be a suitable donor. It has been shown to be a feasible and practical alternative to the conventional multiple ovulation and embryo transfer (MOET) program and it is being more and more used for commercial applications in the world.The Ovum Pick Up technique consists in the transvaginal removal of the oocytes by aspiration of the ovarian follicles with the aid of an ultrasound probe. This procedure is absolutely harmless to the donor. The time taken for the collection is 15-20 minutes during which the donor is contained in a cattle crush.
The oocytes collected are selected in the laboratory and transferred to an incubator in a suitable culture medium to complete the maturation phase. On the day following retrieval, the oocytes are fertilized in vitro with the seed of the chosen bull and kept in incubator for about a week until the stage of embryos suitable for transfer to receiving cows or freezing is reached

Advantage of Ovum Pick UP

• Egg retrieval can be repeated many times twice a week
• Each group of oocytes taken can be fertilized with the seed of a different reproducer. Therefore it is possible to obtain embryo groups with different paternity from the same donor in a very short time, on average 1-2 embryos for picking up from the heifers and 3-4 from the cows
• Ovum Pick Up does not require any preventive hormonal treatment and does not interfere with the normal physiology of the donor;
• Ovum Pick Up embryos can be produced by donors of any age, even during lactation and in the first 3 months of pregnancy without the possible side effects of superovulation: infertility, ovarian cysts, mastitis, traumas related to behavioral changes induced by super dosing hormones and loss of time and milk production.
• For certain donors suffering from long-standing sterility, the Ovum Pick Up can even exert a therapeutic effect by removing the cystic formations tthat are often present on the ovaries of these animals.
• All donors return to estrus 7-10 days after the last withdrawal and can be immediately fertilized.

 

 

Collection and processing of abattoir ovaries:  Ovaries collected from local abattoir in normal saline solution (NSS) having 100 IU/ ml penicillin and 100 microgram/ ml streptomycin sulphate at 37 ⁰C were brought to laboratory and washed with physiological normal saline solution and DPBS (Dulbecco’ s phosphate buffer saline, pH 7.3-7.4) before subjecting to the oocytes recovery.

Recovery/ Collection of oocytes: Different methods are using for recovery/ collection of oocytes with variable success rates.

• Scoring Method: A hemostat is attaching to the base of the ovary to hold the ovary in a place. The excess issue is cut from the ovarian stalk. Ovary is hold above beaker containing oocyte collection medium and 2-3 mm deep incision across all visible follicle (2-6 mm in diameter) are made with sterile surgical blade, with instant rinsing and tapping the ovary to release maximum oocytes.
• Slicing Method: Ovary were placed in a plastic petri dish containing a saline solution and were chopped into small pieces with surgical blade. The cumulus oocyte complexes were selected from the saline solution.
• Puncture Method: Follicle visible on surface ranging from 2-6 mm in diameter were punctured with an 18gauge sterile needle. The cumulus oocyte complexes were selected from the follicular fluid.
• Aspiration method: The follicular fluid from surface follicle (2-6 mm) was aspirated through sterile 18gauge needle attached with 5 ml syringe containing a sterile solution. Aspirated content was expelled into a fresh petri dish containing the oocyte collection media and cumulus oocyte complexes were selected from it.

Oocytes maturation: The oocytes were recovered by puncturing the visible surface of atretic follicle (1-5 mm diameter) with an 18- gauge needle into a 35 mm sterile glass petridish containing oocytes collection media (OCM). The oocytes were graded as excellent, good, fair and poor depending upon the feature of cumulus layers and cytoplasm as per Kharche et al. (2008). Only excellent and good quality oocytes (grade A and B) were subjected to the process of in-vitro maturation.

The selected cumulus oocyte complexes (COCs) were washed 10-20 times in 100 μl drop of oocyte handling media (OHM) followed by 8-10 washing in 100 μl drop of maturation medium (MM) which had been pre equilibrated  in a CO₂ incubator at 38.5 + 1⁰C for 2 h. About 10-15 cumulus oocyte complexes (COCs) were placed in micro drop of 50 μl of the maturation medium (TCM-199, M-7528) containing either 20 % concentration of EGS or 20 % NCS or 10 % NCS plus hormone (FSH @ 1microgram/ ml + LH @ 100 IU/ ml + E₂ @ 1 microgram/ ml) or 20 % CFF which had been equilibrated at 38.5 ⁰C in a CO₂ incubator. The oocytes in the50 μl droplets were covered with sterile mineral oil and cultured in humidified atmosphere of 5 % CO₂ in air at 38.5 + 1⁰C for 27 h in a CO₂ incubator. The maturation of oocytes was confirmed by cumulus expansion and presence of first polar body after 27 h of incubation with maturation media.  The maturation of oocytes were done as per Singh et al. (2009).

In-vitro capacitation

A 50 μl of neat proven semen, with gross motility of + 5 or + 4, was transferred into a centrifuge tube containing 5 ml of fertilization media i.e. fert-TALP. This  was subjected to centrifugation at 1800 rpm for 10 min, after which a clear pellet of spermatozoa was formed. The supernatant was discarded by gentle pipetting without disturbing the semen pellet. The semen pellet was resuspended into 5 ml of fertilization media and the process of centrifugation was repeated. The supernatant was again discarded and the pellet was resuspended in the equal volume of fertilization media. This was then kept for equilibration in a CO₂ incubator for 30-45 min. Following equilibration, the semen sample was again centrifuged (1800 rpm for 10 min) and the supernatant was discarded. The washing was essential to get rid of seminal plasma, impurities and contamination of cell debris etc. After a final washing, 50 μl of the semen pellet was added into 400 μl of pre equilibrated (in a CO₂ incubator for 2 h) fertilization media. Finally, sperm concentration was assessed using Neubaur’s hemocytometer. The in-vitro capacitation was done as per Singh et al. (2010).

In-vitro fertilization of in-vitro matured oocytes

After 27 h of culture, oocytes were evaluated for cumulus expansion and presence of first polar body. Only the mature oocytes were picked up and denuded by treating them with 0.1 % hyaluronidase and by repeated pipetting through a fine boar pipette. The denuded oocytes were washed 8-10 times in a fert-TALP medium which had been pre equilibrated in a CO₂ incubator at 38.5+1⁰C for 2 h and then transferred into 25 μl drops of fert-TALP medium under sterile mineral oil, equilibrated for 2h at 38.5+1⁰C in a CO₂ incubator. The capacitated semen having final concentration of 1 x 10⁶ live sperm per ml, of this 50 μl was used for in-vitro fertilization into each fertilization drop containing mature oocytes with a denuded cumulus cell mass. Oocytes were co-incubated with spermatozoa for 18 h at 38.5+1⁰C in an atmosphere of 5 % CO₂ in a humidified air. The in-vitro fertilization of in-vitro matured oocytes was done as per Singh et al. (2009).

In-vitro culture of in-vitro fertilized oocytes

Eighteen hours post insemination, oocytes were washed in a culture media (Embryo development medium, EDM) 8-10 times in order to separate adhering sperm cell. The oocytes were finally transferred either on monolayers (equilibrated with EDM under oil for 18 h) or 50 μl drop of EDM co-incubated with oviductal epithelial cells under sterile mineral oil (equilibrated for 2 h at 38.5+1⁰C in a CO₂ incubator) for 48 h in a drop. They were cultured at 38.5+1⁰C at 5 % CO₂ in a CO₂ incubator for 7 days. The in-vitro culture of in-vitro fertilized oocytes was done as per Singh et al. (2010).

Assessment of cleavage rate and embryo development

            Cleavage rate was evaluated 48 hour post insemination, by counting the number of cleaved ≥ 2 cell embryos/ number of oocytes used for fertilization. After evaluation of cleavage rate, embryo development was seen morphologically every 24 h under stereozoom microscope and classified  as 2 cell, 4 cell, 8 cell and > 8 cell embryos depending on a possible in-vitro cellular block. After every 24 h, 50 % media was replaced with a fresh pre-equilibrated EDM media.

Oocyte/embryo cryopreservation and vitrification

Continuous availability of viable, developmentally competent oocytes  and/or embryos has been critical  to  recent  progress in IVP because of the relatively short fertile life span of mammalian oocytes and/or embryos. Hence, storage of unfertilized oocytes would generate a readily available source, which allow the experiments to be carried out  at  convenient  time  andcould  therefore  be  of  practical  importance  for establishment of gamete bank, from which particular genetic combinations could be derived.  During  the  past  few  decades,  significant  progress  in  cryopreservation  of mammalian  oocytes  and embryos has  been achieved. Live offspring of at least  25 species  resulted  from  transfer  of  cryopreserved  embryos  or  oocytes  have  been successfully produced (Gajda and Smorg, 2009).  The  major  problem  associated  with  cryopreservation  of  germplasm  is mechanical as well as osmotic damage during processing steps. With the advent of glycerol, which is a cryoprotecting (CP) agent, cryopreservation process became more feasible. Other CPs is also in widespread use, alone or in various combinations. These include permeating CPs such as ethylene glycol and propylene glycol (Chen  et al., 2005; Luz et al., 2009) and non-permeating ones such as sucrose, glucose, or fructose (Barcelo-Fimbres and Seidel, 2007).  With the development in traditional cryopreservation methods, vitrification of germplasm was introduced (Rall and Fahy, 1985). Vitrification is a simple, faster, less expensive technology  than  slow  freezing.  Moreover,  it  was  shown  to  be  more effective than slow freezing for material more sensitive to chilling (Vajta et al., 1996). Cryopreservation  of  oocytes  by  vitrification  was  tested  with  variable  success  for oocytes of bovine (Hochi et al., 2000), swine (Huang and Holtz, 2002), equine (Hurt et al., 2000) and buffalo (Sharma and Loganathasamy, 2007).  Freezing of embryo is an established commercial practice and the major aim of it is to preserve  the embryo in a viable condition from which it  may be revived to continuea normal development and avoid ice crystal formation that can be harmful for  embryo  when thawing.  Embryo  freezing  is  an  essential  component  in  the commercial embryo transfer (ET) programs as there is an obvious need to store the embryos on temporary basis until they are transferred, as the embryo viability starts to decline after  12  h of storage in holding  media. With regard to the embryo sexing, naturally, both male and female embryos should be stored to ensure representation of both sexes and wide genetic diversity. Preservation of oocytes reduces the risk and expense involved in transport of live  animals,  hazards  of  disease  transmission  and  also provides  insurance  against catastrophes  and  natural  disasters.  On the  other hand,  Preservation of  oocytes and embryos  of  endangered  species  safeguards  from  danger  of  extinction. Cryopreservation can be considered as a useful conservation strategy for endangered breeds. Fo these species,  cryobanking  of  embryos could be helpful  in  establishing founder populations with the aim of eventual reintroduction into the wild (Ptak et al., 2002).

       Zygote Intra-Fallopian  Transfer (ZIFT)  and Gamete  Intra-Fallopian Transfer (GIFT) ZygoteIntra-Fallopian  Transfer (ZIFT), also referred  to  as Tubal Embryo Transfer (TET), is an ART technique in which embryos are transfered into the fallopian tubes for  purposes of  achieving pregnancy.  Meanwhile,  Gamete Intra-Fallopian  Transfer (GIFT) allows the transfer of gamete into the fallopian tubes. The obvious advantage of ZIFT over GIFT is that as in IVF, it is possible to document fertilization. On the other hand, both ZIFT and GIFT procedures require the female to have at least one functioning fallopian tube which is a disadvantage when compared with IVF. These different techniques are used to achieve pregnancy in high genetically merit animal with reproduction  problems  or  to  achieve  implantation  of  produced  embryos  in surrogate mothers.

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K.P. Singh*¹, Bhoopendra Singh² and Praneeta Singh³

Government Veterinary Hospital, Deoranian, Bareilly,

Department of Animal Husbandry, Uttar Pradesh, India

 

1: Veterinary Officer, Government Veterinary Hospital, Deoranian, Bareilly, Uttar Pradesh Email: drkpsvet@rediffmail.com

2: Assistant Professor, Department of Animal Reproduction, Gynaecology and Obstetrics, C.V.Sc.&A.H, ANDUAT, Ayodhya, Uttar Pradesh Email:drbsvet@gmail.com

3: Assistant Professor, Department of Livestock Products Technology, C.V.A.Sc., GBPUAT, Pantnagar, U.S.Nagar, Uttrakhand Email: vet_praneeta12@rediffmail.com

*Corresponding Author: Veterinary Officer, Government Veterinary Hospital, Deoranian, Bareilly, Uttar Pradesh Email: drkpsvet@rediffmail.com

 

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