Cryoconservation du sperme et des embryons chez les petits ruminants

Résumé

La congélation du sperme et des embryons est une biotechnologie importante pour la conservation et propagation de la génétique. L’objectif de cet article est discuter les techniques de congélation établies ainsi que les acquisitions récentes dans ce domaine chez les ovins, caprins et petits ruminant sauvages. L’utilisation du sperme congelé en production est plus développée chez les caprins que chez les ovins. Ceci est principalement dû au besoin de l’utilisation de l’insémination par laparoscopie chez les brebis. La plus grande différence entre les caprins et les ovins est le comportement du plasma séminal vis as vis des protéines du jaune d’œuf. Récemment, les efforts de recherches se sont penchés principalement sur le développement de nouvelles approches pour l’amélioration des dilueurs de congélation. Les axes les plus importants étant le remplacement des protéines animales (jaune d’œuf et lait) par les lécithines végétales, l’utilisation de cryoprotcteurs autres que le glycérol, et la réduction des effets de l’oxydation des lipides. L’introduction de méthodes d’analyses de la morphologie et fonction des spermatozoïdes, telles que les techniques de fluorescence, les tests hypopsmotiques et l’évaluation numérique de la mobilité, permet une évaluation plus rigoureuse des effets de ces facteurs sur la congélabilité du sperme La conservation des embryons des petits ruminants par vitrification se développe de plus en plus et pourrait remplacer les techniques lentes, plus laborieuses et coûteuses. Chez les ovins, les morulas et blastocystes semblent être plus résistants à la congélation. Par contre chez les caprins, les blastocystes en expansion et éclos sont plus résistants à la congélation que les morulas et jeunes blastocyste. Les taux de gestation obtenus après transfert d’embryons congelés sont très intéressants surtout quand la gestion des receveuses est adéquate et les techniques de transfert sont maîtrisées.

Mots-clés: Congélation, sperme, embryons, insémination, fertilité, dilueurs.

INTRODUCTION

Cryopreservation of sperm and embryos of small ruminants is an important reproductive biotechnology for the preservation of rare or local breeds and the propagation of genetic material from imported high performing individual animals. Another advantage of these technologies is the introduction in a flock or herd of new genetics without breaching biosecurity measures if donor animals are submitted to rigorous veterinary inspection and disease testing. The objective of this paper is to review the general principles of sperm and embryo cryopreservation and discuss new research aimed at improvement of these technologies in small ruminants.

SEMEN CRYOPRESERVATION

Although sperm preservation is considered common practice in both sheep and goat, new improvements have been introduced mostly to simplify the technique for field use. Improvement in post-thaw quality aims to bypass laparoscopic insemination particularly in sheep. Advances in cryopreservation of small ruminant sperm include development of new extenders to eliminate animal products such as milk or egg yolk. Differences in sperm cryopreservation exist between sheep and goat. Goat sperm is known for the toxic reaction to high levels of egg yolk in the extender.

Cryopreservation of ejaculated sperm

One of the primary conditions for successful cryopreservation of sperm is the initial quality of the ejaculate. Rams and bucks that are candidates for semen cryopreservation and use in artificial insemination programs should undergo a thorough breeding soundness examination (BSE) and disease testing prior to enrollment. Cryopreservation causes biological and functional changes that can affect sperm viability and fertility. These changes occur primarily at the membrane levels and are due to ice crystal formation and changes in membrane permeability. A significant proportion (20 to 30%) of cryopreserved spermatozoa lose their fertilizing ability after thawing. Therefore, only ejaculates of excellent quality (>90% motility and >70% normal morphology) should be used for cryopreservation (Dorado et al., 2010). Principles for BSE in rams and bucks are described in detail elsewhere in the present publication (Tibary et al., 2018). It is important to take into account seasonal effects on initial quality of the ejaculate and seminal plasma proteins and their interaction with membrane stability and ability to withstand freezing and thawing (Goularte et al., 2014; Sobrinho et al., 2014; Wang et al., 2015). Melatonin treatment of bucks improved fresh semen quality but not cryopreservation (Gallego-Calvo et al., 2015).

Cryopreservation of small ruminant sperm has been practiced for decades. Several extenders have been developed and are either egg yolk (EY) or skim milk based (Table 1) (Cseh et al., 2012; Kucuk et al., 2014; Emamverdi et al., 2015). The main buffers used are citric acid and Tris. Zwitterionic buffer have also been used successfully for small ruminant semen preservation (Tuli and Holtz, 1992). Egg yolk low-density lipoproteins (lecithin and phosohatidylcholines) protect the sperm membrane during the freezing and thawing processes. A major difference between ram and buck semen is their behavior toward egg yolk, which tends to coagulate in presence of seminal plasma from bucks. This effect has been attributed to the presence of a specific lipase belonging to the pancreatic lipase-related protein 2 family that is secreted by the bulbourethral glands (egg yolk coagulating enzyme or BUSgp60) (Cseh et al., 2012). Goat ejaculates are washed by centrifugation (700 x g for 15 minutes) in PBS or extender without egg yolk prior to dilution with egg yolk containing extenders (Batista et al., 2014). Alternately, semen can be diluted with extenders containing very low level of egg yolk (2 to 3%) (Shamsuddin et al., 2000; Bispo et al., 2011; Anand and Yadav, 2016). Other researchers have used up to 12% egg yolk without washing (Cabrera et al., 2005).

Following dilution, semen is slowly cooled to 5°C for a minimum of 1.5 to 2 hours, loaded in straws and frozen in liquid nitrogen vapors (Cseh et al., 2012). The final dilution of semen prior to cryopreservation is generally between 100 and 200 million sperm per mL. Increasing the concentration above 400 million per mL results in a decrease in post-thaw quality (Alvarez et al., 2012a).

There are few studies on the optimal freezing rate for ram and buck sperm. The optimal freezing rate should avoid intracellular ice formation, cell shrinkage and long exposure to high osmotic and ionic strength. Freezing rates commonly used for small ruminant sperm cryopreservation range from -10 to -100°C/min. In a recent study on ram semen, the best freezing rate was determined to be -40°C/minute when semen was diluted in Tris-glucose EY and cooled at 4°C for 2 hours in 0.25 mL straws (Fang et al., 2016). Although protocols for cryopreservation of ram and goat spermatozoa have been successfully used for several decades, introduction of technologies such as sex-sorted semen requires improvement of cryopreservation (Bathgate et al., 2013; Quan et al., 2015). Factors that have been studied recently include the type and method of addition of cryoprotectant (Silva et al., 2012b; Pelufo et al., 2015), cooling rate and equilibration time (Ahmad et al., 2015) and individual male variability. Cryoprotectants are generally added in a single step or in two steps (after cooling). Cooling rates and equilibration times from 2 to 8 hours have been tested (Ahmad et al., 2015). Equilibration time of 2 hours seems to be sufficient for most protocols (da Silva et al., 2014). Equilibrated semen is placed in 0.25 or 0.5 mL straws and cooled at a rate of 3°C/min from 5°C to -8°C and at 25°C /minute from -8 to -120°C.

Individual male variation exists in the ability of sperm to withstand freezing. This variation may be linked to differences in protein content of seminal plasma and membrane stability (Moura et al., 2010; Romon et al., 2013). These individual variations are illustrated by difference in changes in sperm head volume during the freezing process (Romon et al., 2013). Genetic variation may also affect cryopreservation of semen. Recently, the heat shock protein 70 (HSP70) gene has been suspected to have an effect on semen quality and possibly freezing ability in Boer goats under tropical conditions (Nikbin et al., 2014).

Table 1: Extenders used for cryopreservation of small ruminant sperm

New developments in sperm cryobiology

Replacement of egg yolk

As stated above, addition of egg yolk to extenders provides cryoprotection to the sperm through its low-density lipoprotein. Egg yolk from different species has been tested resulting in some differences (Kulaksiz et al., 2010). Egg yolk, as a membrane protective agent in semen extenders, has become progressively undesirable because of its antigenic proprieties (Ustuner et al., 2014) and the reluctance of some countries to import semen frozen with egg yolk containing extenders for biosecurity reasons. This led to its replacement by lecithins from vegetal origin such as soybean (Del Valle et al., 2013; Emamverdi et al., 2013; Vidal et al., 2013; Salmani et al., 2014; Bohlool et al., 2015; Chelucci et al., 2015; Sharafi et al., 2015; Masoudi et al., 2016a; Toker et al., 2016). The combination of soy milk and glycerol has been used successfully for cryopreservation of ram sperm (Jerez et al., 2016). The use of soy extender (Bioxcell®) for goat semen has been shown to remove the need for washing of ejaculates prior to dilution and freezing (Sariozkan et al., 2010; Roof et al., 2012).

Addition of antioxidants

Sperm membrane has a high concentration of polyunsaturated fatty acids and is extremely sensitive to oxidative stress during cryopreservation (Del Olmo et al., 2015; Amidi et al., 2016; Toker et al., 2016). Research has focused, in the last decade, on improvement in cryopreservation of ram and buck semen through addition of various antioxidants to extenders with variable results. Enzymatic antioxidants such as glutathione (GSH), superoxide dismutase (SOD) and catalase (Silva et al., 2011; Santiani et al., 2014) have been used in various concentrations to improve post-thaw sperm quality. In rams, addition of SOD, GSH and MNTBAP (a superoxide-dismutase mimetic) seems to reduce acrosomal damage (see review by Budai et al., 2014). Addition of Trolox and catalase to tris-egg yolk extender significantly reduced lipid-peroxidation in ram semen (Maia et al., 2010; Sicherle et al., 2011; Silva et al., 2013; Camara et al., 2016). Trolox was also beneficial for preservation of sperm of plasma membrane and mitochondrial sheath integrity of goat sperm frozen in a skim milk based extender (Soares et al., 2015).

Other antioxidants (zinc, tocopherol, albumin, glutathione, taurine, hypotaurine, carnitine, carotenoids, cysteine, urate and prostasomes) (Mata-Campuzano et al., 2011; Mata-Campuzano et al., 2012; Budai et al., 2014; Mata-Campuzano et al., 2015) have also been tested in small ruminant sperm cryopreservation with variable results (Budai et al., 2014). Oxidized gluthatione (Camara et al., 2011; Razliqi et al., 2015; Hazarika et al., 2016), cysteamine (Najafi et al., 2014a; Alcay et al., 2016), methionine (Tuncer et al., 2010; Alcay et al., 2016), catalase, cysteamine (Bucak et al., 2007), Butylated hydroxytoluene (BHT) (Naijian et al., 2013; Alcay et al., 2016), ergothioneine (Coyan et al., 2011; Najafi et al., 2014a), hypotaurine (Bucak et al., 2013), taurine (Bucak et al., 2007) and trehalose (Bucak et al., 2007; Tuncer et al., 2010; Najafi et al., 2013; Tuncer et al., 2013; Bohlool et al., 2015) have all been used as antioxidants and reactive oxygen species scavengers. Resveratrol and quercetin have also been shown to improve post-thaw viability and mitochondrial membrane potential (Silva et al., 2012a). Dithiothreitol, a protamine disulfide bond reducing agent, has been shown to have a positive effect on post-thaw motility of ram semen (Baspinar et al., 2011). All these approaches proved to be beneficial in increasing viability, post-thaw motility and acrosome integrity. However, the true effect of some these antioxidants on sperm viability and post-thaw fertility remains controversial. In one study, addition of cysteamine to electroejaculated ram semen was detrimental to cryosurvival while addition of iodixanol was beneficial (Cirit et al., 2013). Glutathione addition had no beneficial effect on buck semen extended in soybean lecithin containing extender (Salmani et al., 2013). In one study on ram semen, no advantage was found with addition of gluthatione, superoxide dismutase or catalase (Camara et al., 2011). Supplementation of extender with vitamin C has been shown to improve post-thaw buck sperm quality (Gangwar et al., 2015). Other studies have shown little benefit or negative effect of some antioxidants on post thaw sperm quality (Yildiz et al., 2015; Zhandi and Sharafi, 2015). The effects of antioxidants on post-thaw sperm quality merits however further studies to elucidate the interaction between specific extender and concentration of antioxidants (Mata-Campuzano et al., 2015; Zhandi and Sharafi, 2015).

Another approach to reduce reactive oxygen species due lipid peroxidation in semen is through nutrition. Addition of fish oil to ram diet has been shown to improve sperm quality and in vitro fertilization results (Behzad et al., 2014; Esmaeili et al., 2014; Masoudi et al., 2016b). Semen quality was also shown to be improved in rams supplemented orally with vitamins E and C (Memon et al., 2013; Cofre-Narbona et al., 2016). Dietary supplementation with n-3 polyunsaturated fatty acid of rams had limited effects on the quality of liquid stored semen (Towhidi et al., 2013; Fair et al., 2014).

Alternative cryoprotectants

Glycerol is the most commonly used cryoprotectant for sperm. However, exposure to high levels of glycerol is toxic and can be detrimental to sperm function and viability in utero. Therefore, research efforts have focused on alternative cryoprotectants. In goats, dimethylsulfoxide (DMSO) and ethylene glycol (EG) did not present any added advantage in sperm cryopreservation (Buyukleblebici et al., 2014). In rams, EG at concentrations of 3 or 5% was beneficial but not as good as glycerol (Silva et al., 2012b). DMSO was ineffective in ram semen preservation (Bezerra et al., 2011; Moustacas et al., 2011). In rams, propanediol, sucrose and trehalose did not perform as well as glycerol (Nur et al., 2010). However, trehalose supplementation to extenders improved cryopreservation of Boer semen in Tris-citric acid extender (Naing et al., 2010). Amino acids (L-glutamine and L-proline) have been used successfully to improve cryoprotection and reduce lipid peroxidation (Farshad and Hosseini, 2013; Mehr and Noori, 2013; Sangeeta et al., 2015). Addition of cholesterol-loaded cyclodextrin may allow reduction of the concentration of glycerol to 3% (Moce et al., 2010; Awad, 2011; Konyali et al., 2013; Moce et al., 2014; Motamedi-Mojdehi et al., 2014).

Counteracting capacitation-like changes

One of the major effects of cryopreservation on sperm is induction of capacitation-like changes (i.e. cryocapacitation). Several studies have shown that semen collected by electroejaculation (i.e. higher seminal plasma content) has better cryotolerance than semen collected by artificial vagina (Jimenez-Rabadan et al., 2013; Ledesma et al., 2015). However other studies did not show any difference between the two methods of collection while others have shown that AV collected semen from bucks has better post-thaw motility (Jimenez-Rabadan et al., 2012). Addition of seminal plasma to cryopreserved sperm was found to improve fertility in ewes inseminated transcervically (Maxwell et al., 1999). Some proteins that are provided by seminal plasma such as spermadhesins and SPINK3 (Serine Protease Inhibitor Kazal Type 3 or caltrin) interfere with the capacitation pathways by either stabilizing the membrane or inhibiting calcium transport (Maxwell et al., 2007; Zalazar et al., 2016). SPINK3 was found in high concentration in seminal plasma from rams with good freezing ability spermatozoa (Rickard et al., 2016). This protein is known to modulate calcium increase and to downregulate the signal transduction pathway leading to capacitation. Addition of heterologous recombinant SPINK3 to the freezing extender or after thawing was shown to improve post-thaw progressive motility and reduce acrosomal loss (Zalazar et al., 2016). Post-thaw addition of seminal plasma was also shown to reverse cryopreservation damage (Bernardini et al., 2011). There is a seasonal variation in the protective effect of seminal plasma on frozen-thawed ram spermatozoa (Leahy et al., 2010). Despite these studies, the effect of addition of seminal plasma to cryopreserved sperm remains controversial (Prado et al., 2013; Rovegno et al., 2013).

Other additives

Fruits juices from pineapple and orange (Daramola et al., 2016b), coconut water (Daramola et al., 2016a), honey (Jerez-Ebensperger et al., 2015) as well as herbal antioxidants (Mascaro et al., 2013; Zanganeh et al., 2013; Baghshahi et al., 2014) have been used with various degrees of success.

Cryopreservation of semen from other small ruminants

Commercial extenders developed for the cryopreservation of ram and buck semen have been used successfully for the cryopreservation of semen from farmed cervids (i.e. elk, white-tailed deer) and wild small ruminants (i.e. Bighorn sheep, roe deer, mouflon...). Semen is generally collected by electroejaculation with or without ultrasound guided rectal massage under general anesthesia. In farmed white tail deer, soybean (Andromed®) and liposome-based (Optixcell®) extenders were found to be superior to Tris-based extenders (Biladyl® or Triladyl®) (Stewart et al., 2016). Electroejaculated semen from captive roe deer (Capreolus capreolus) showed better post-thaw motility and acrosome integrity when from frozen in Berliner Cryomedium® (TES-Tris-fructose-lactose-EY) compared to Tris-citric acid-glucose or TES-Tris-glucose (Prieto-Pablos et al., 2016). Similar extenders have been used successfully for the cryopreservation of semen from endangered gazelles (Gazella cuvieri, G. dama mhorr, and G. dorcas neglecta) (Garde et al., 2003; Garde et al., 2008), ibex (Capra pyrenaica) (Santiago-Moreno et al., 2009b; Coloma et al., 2010a; Coloma et al., 2010b; Coloma et al., 2011; Pradiee et al., 2015), chamois (Rupicapra pyrenaica), mouflon (Ovis musimon) and aoudad (Ammotragus lervia sahariensis) (Santiago-Moreno et al., 2013; Pradiee et al., 2016). In our laboratory, electroejaculated semen from Bighorn sheep (Ovis canadensis) has been successfully cryopreserved in ram semen extenders and used for artificial insemination (Rodriguez et al., 2009; Subramaniam et al., 2014).

Cryopreservation of epididymal sperm

Post-mortem cryopreservation of cauda epididymis sperm and its use for artificial insemination is an important area of research. Optimization of the technique allows salvage of genetics from terminal animals or from rare domestic and wild small ruminants (Vasquez et al., 2013). Epididymal sperm can be harvested from severely injured valuable males or wildlife and cryopreserved. Testes can be cooled to 5°C and stored for up to 48 hours before processing (Turri et al., 2014). This technique has been used successfully in a variety of species including the ibex (Santiago-Moreno et al., 2006a; Santiago-Moreno et al., 2006b; Santiago-Moreno et al., 2007; Santiago-Moreno et al., 2008; Santiago-Moreno et al., 2009a; Fernández-Santos et al., 2011; Pradiee et al., 2014; López-Saucedo et al., 2015), Iberian red deer, roe deer, catabrian chamois (Martinez-Pastor et al., 2005; Martínez et al., 2008), and Bighorn sheep (Rodriguez et al., 2010; Hansen et al., 2012; Campbell et al., 2014) (Figure 1).

Advances in method of evaluation of cryopreserved sperm and relationships with fertility

Method to evaluate post-thaw semen quality has been a subject of research for several decades. Post-thaw progressive motility is often used as the primary criterion. Velocity and linearity determined by computer assisted sperm analysis (CASA) seems to be a more reliable indicator of quality and correlates with fertility (Furstoss et al., 2010; Del Olmo et al., 2013). In addition to motility, evaluation of membrane integrity, viability and functional tests have become more and more commonly used to objectively evaluate sperm quality. Viability or membrane integrity can be determined with eosin-nigrosin staining (Rekha et al., 2016). However, fluorescence techniques such as SYBR-14 and prodium iodide offer a more repeatable measure. SYBR-14 is used as a membrane-permeable DNA intercalating agent with maximum emission of 516 nm (green) and readily stains all nuclei. Propidium iodide is used as an intercalating agent with maximum emission of 617 nm (red), but it only stains the nucleus if the sperm plasma membrane is damaged which makes it a useful counter stain for dead cells (Yaniz et al., 2013; Stewart et al., 2016) (Figure 2). Membrane integrity can also be evaluated with carboxyflurescien diacetate and propidium iodide (Soares et al., 2015).

Membrane integrity can also be assessed by the hypo-osmotic swelling test (HOST). The HOST solution is prepared using a mixture of fructose (9 g/L) and sodium citrate (4.9 g/L) (Alcay et al., 2016; Quan et al., 2016; Rekha et al., 2016) or a 100 mOsm solution of sucrose. Semen (20 µl) is added to the solution (200 µl) and incubated for 30 to 60 minutes at 37ºC. Spermatozoa (200) are evaluated under the microscope (400×). Sperm with swollen membranes (curled tails) are considered to have an intact membrane (Figure 3).

Figure 2: SBYR-14 and propidium iodide viability staining: Red sperm (stained with PI) have damaged plasma membrane, Green sperm (SYBR-14 stained are viable).

Acrosome status can be assessed with Chlortetracyclin staining (Najafi et al., 2014b) or by a combination of Fluorescein isothiocyanate-conjugate peanut (Pisum sativum) agglutinin (FITC-PNA) and PI. This stain has a maximum emission of 521 nm (green) and targets the inner leaflet of the outer acrosomal membrane identifying damages acrosomes. PI is used as a counterstain to allow for simultaneous sperm viability and condition of acrosome (Soares et al., 2015; Alcay et al., 2016; Stewart et al., 2016) (Figure 4).

Figure 3: Hypoosmotic swelling test: sperm with damaged membranes do not swell (straight), normal sperm shows various coiling of the tail and mid-piece due to swelling.

Figure 4: FITC-PNA damaged acrosome (green fluorescence).

Sperm mitochondrial status can be assessed using mitochondrial stain rhodamine l23 or 5,5′,6,6′tetrachloro-1,1′,3,3′-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1). For staining, 300 μL of semen is mixed with 2.5 μL of 1.53 mM solution of JC-1 in DMSO and 2.5 μL of 0.2 % solution of PI in distilled water. The sample is gently mixed and incubated at 37°C in dark for 20 minutes. JC-1 aggregates inside the functional mitochondria. Highly functional mitochondria are more penetrable by the stain and glow yellow-orange compared to green fluorescing weak functioning mitochondria (Figure 5).

Figure 5: Mitochondrial activity assessed with JC-1. Sperm with midpiece yellow-orange fluorescence have highly functional mitochondria. Green fluorescence denotes weak activity

Another sperm quality test that has gained a lot of interest is sperm chromatin structure assay (SCSA) using acridine orange stained sample analyzed by flow cytometry under 488 nm argon excitation laser. Acridine orange is a cell permeant nucleic acid binding dye that emits green fluorescence when bound to double stranded DNA (normal) and red when bound to single stranded DNA (abnormal) (Stewart et al., 2016).

Sperm death can be determined using apoptosis detection kits. One such kits uses PI and Annexin V. PI can only penetrate membrane of necrotic cells to stain nucleus. PI positive population, dead; Annexin V-FITC positive and PI negative population, early apoptotic; both Annexin V-FITC and PI positive population, late apoptotic; both Annexin V-FITC and PI negative population, normal (Quan et al., 2016). In situ cell death detection can also be determined enzymatically using a Terminal deoxynucleotydyl transferease dUTP nick end labeling (TUNEL) assay to detect DNA fragmentation (Alcay et al., 2016). Apoptotic cells can also be identified by mitochondrial membrane potential using specific stains such as MitoLight® (5,5_,6,6_-tetrachloro-1,1_,3,3_-tetraethylbenzimidazolylcarbocyanine chloride) that differentially stains mitochondria of healthy cells red while it remains as monomers in the cytoplasm and fluoresces green (Quan et al., 2016).

FERTILITY

Pregnancy rates using frozen-thawed semen vary from 50 to 70% in goats. Several factors, including breed, season, male, dose of semen, method of synchronization and flock/herd management (Anel et al., 2005; Arrebola et al., 2014). Artificial insemination is performed at a fixed time following synchronization or 12 hours after onset of estrus. In goats, most breeds can be inseminated by transcervical catheterization except for very small breeds where laparoscopic insemination is required in order to deposit semen in utero (Nordstoga et al., 2010b).

In sheep, the main challenge in artificial insemination with frozen-thawed semen has been the difficulty in placing semen in utero via transcervical catheterization because of the morphology of the cervix (Moura et al., 2011; Richardson et al., 2011; Robinson et al., 2011). Traditionally, ewes are inseminated with frozen-thawed seem via laparoscopy (Masoudi et al., 2017). Laparoscopic insemination does not seem to be affected by side of insemination (ipsilateral vs contralateral to ovulation) (Anakkul et al., 2014). In recent years, transcervical or vaginal inseminations gained more popularity. Several treatments have been tested to improve pregnancy rate following intracervical insemination. These include the use of estrogens, relaxin, oxytocin (IM) (Bartlewski and Candappa, 2015), topical application of PGE2 on the cervix (Horta et al., 2010; Barbas et al., 2013; Bartlewski and Candappa, 2015) and treatment with carazolol (a beta adrenergic blocking agent) (Gündüz et al., 2010). However, these treatments result in only modest improvement of pregnancy rates or sometimes a reduced fertility. Deposition of semen in the vaginal fornix can result in pregnancy although the overall conception rates are generally 10 to 20% lower than with intracervical insemination (Richardson et al., 2012a). A recent small study on vaginal insemination using a dose of 400 million spermatozoa per insemination resulted in a 50 % conception rate (Richardson et al., 2012a). Despite a reduced pregnancy rate with vaginal insemination, the technique is more attractive given the lesser cost and animal welfare issues compared to laparoscopic insemination (Paulenz et al., 2007). However, excellent semen quality is required with vaginal insemination (Paulenz et al., 2007; Richardson et al., 2012b). In sheep, epididymal sperm produced equal pregnancy rates as semen collected with artificial vagina. However, semen collected by electroejaculation produced lower pregnancy rates (Alvarez et al., 2012b).

CRYOPRESERVATION OF EMBRYOS

Embryo cryopreservation has been used in small ruminants since the late 1970’s. There are two methods of embryo cryopreservation; the conventional slow method and vitrification (Massip, 2001). The first step in embryo cryopreservation is selection of excellent quality embryos (Grade I or II) (Dalcin et al., 2013) (Figure 6). The main recent advances are in the development of simple procedures of vitrification making the process cheaper and faster for field use.

Cryopreservation of embryos by the slow cooling methods

The conventional method for embryo cryopreservation is often referred to as a slow-freezing method because it involves several steps which include, dehydration and equilibration with cryoprotectant, slow cooling to the seeding point, seeding and holding then further cooling at a slow rate prior to plunging the embryos in liquid nitrogen (Youngs, 2011).

Cryoprotectant addition and equilibration

The cryoprotectant can be added in a single step or in multiple steps with increasing concentration. Embryos are equilibrated in each solution for 5 to 10 minutes depending on the cryoprotectant used. The most common cryoprotectants used are glycerol of ethylene glycol. After equilibration, the embryos (2 per straw) are loaded in 0.25 mL straws (Youngs, 2011).

Initial cooling and seeding

The slow embryo freezing methods requires the use of a controlled rate biological freezer (Figure 7). These devices are designed to cool the embryos at a rate of 1°C/min from room temperature to the seeding point (-6 to -6.5°C). Seeding is the process of induction of ice crystal formation. This is achieved by touching the meniscus of the medium in the straw with very cold small tweezers or cotton-tipped stick that have been placed in liquid nitrogen (Figure 8). The formation of ice crystal in the medium surrounding the embryo results in further dehydration of the embryo. The straw is held at the seeding temperature for approximately 10 minutes before resuming the cooling process (Youngs, 2011).

Final cooling and storage in liquid nitrogen

After holding at seeding temperature, the straws must be cooled at a rate of 0.5°C/min to approximately -34 or -35°C where they are held for approximately 10 minutes prior to direct plunging in liquid nitrogen (-196°C) then transferred to a liquid nitrogen tank for storage (Youngs, 2011).

Thawing of embryos frozen with the conventional method

Cryopreserved embryos must be thawed following a strictly defined method. Straws are usually removed from the liquid nitrogen tank and held at room temperature for 3 to 5 seconds. This would reduce the risk of cracking the zona pellucida and explosion of the straws. Straws are then placed in a water bath at 35 to 38°C for 25 to 30 seconds. The embryos are removed from the straw and placed in a rehydration (cryoprotectant removal) medium prior to transfer. The process of cryoprotectant removal or rehydration is performed in a stepwise or single step fashion. The stepwise technique requires embryos to be placed successively in media with lower concentrations of cryoprotectant (1.5, 1.0 and 0.5 Molar) for 10 minutes per step. In the single step technique, the embryos are placed in a 1.0 Molar solution of sucrose (a non-penetrating sugar) for 10 minutes (Youngs, 2011).

In recent years, the trend has been to freeze embryos using the direct transfer method which does not require removing the embryos from the straws. The direct transfer method requires the use of ethylene glycol as the cryoprotectant. Because ethylene glycol is a small molecule, it diffuses quickly out of the embryo upon transfer to the recipient. It is very important to minimize the time from thawing to transfer into the uterus when using this method. In sheep, a one-step addition of 1.5 M ethylene glycol followed by the one step removal in 1 M sucrose after controlled freezing and thawing yielded very high survival rate (>70%) (McGinnis et al., 1993).

In goats, the maximum survival of frozen-thawed embryos is attained with expanded, hatching or hatched blastocysts (Li et al., 1990) (Figure 8).

Embryo vitrification

Vitrification is an ultrafast fast method for freezing (2500°C/min) resulting in “glass” (i.e. vitrous state) instead of ice crystal formation. The technique was first described for mammalian embryos in 1985 (Rall and Fahy, 1985). This avoidance of intracellular and extracellular ice crystal formation reduces cellular damage and improves embryo viability (Depaz et al., 1994). In addition, this technique does not require any sophisticated equipment, which reduces the cost of cryopreservation of embryos. Vitrification uses media containing high concentration of permeating cryoprotective agents. Embryos are placed in a first medium of 2 Molar of the cryoprotectant. Following equilibration, the embryos are placed immediately in a medium with much higher concentration of cryoprotectant (e.g 7 Molar) for a very short period of time (30 to 45 second) then they are plunged in liquid nitrogen (Massip, 2001). Several vitrification protocols have been reported for sheep and goat embryos. There seems to be an important difference between species in terms of behavior of embryos towards the vitrification medium (goats (El-Gayar and Holtz, 2001; Al Ahmad et al., 2012; Araujo-Lemos et al., 2015); sheep (Ali and Shelton, 1993; Bettencourt et al., 2009; de Araujo-Lemos et al., 2014; Santos Neto et al., 2015)).

In sheep, vitrification of embryos was reported to be as efficient as conventional freezing (Depaz et al., 1994; Martinez and Matkovic, 1998; Baril et al., 2001; Isachenko et al., 2003; Bettencourt et al., 2009; Green et al., 2009). Several improvements of the vitrification protocol have been achieved in recent years resulting in improved viability of embryos. There is a general agreement amongst authors that ovine embryos are more resistant to cryodamage than those of other species (Bettencourt et al., 2009).

A recently described protocol resulted in good survival of embryos. Embryos are placed in a basal solution of Hepes-TCM 199 supplemented with 20% Fetal Bovine Serum (FBS) for 5 minutes then transferred into a medium containing 10 % ethylene glycol (EG) and 10 % dimethylformamide (DMF) for 1 minute and moved to a solution of 20 % EG, 20% DMF and 0.5M sucrose and frozen using the open pulled straw technique by plunging in liquid nitrogen (Araujo-Lemos et al., 2015).

Sheep and goat embryos have been vitrified successfully using a standard simple commercial protocol used for bovine embryo vitrification. Embryos are placed in a commercial holding medium (Syngro®) until vitrification. The commercial vitrification protocol (Bovipro®, Minitube, Germany) consisted of a basic medium supplemented with 20 % FBS. The embryos are placed for 5 minutes in BM+10% glycerol then transferred into BM + 20 % glycerol +20 % EG for 5 minutes. Finally, the embryos are placed in the vitrification solution (BM+25 % glycerol+ 25 % EG) for 30 seconds. Embryos are loaded in 1 µl of vitrification medium in the tip of a micropipette and then placed in a 3.6 ml cryotube filled with liquid nitrogen. For thawing, the micropipette tips are warmed between the thumb and the middle finger for 10 seconds and the embryos are immersed in BM at 25°C in three dilution steps for 5 minutes each (12.5 % G+12.5 % EG+ 0.5 M sucrose, 0.5 M sucrose and 0.25 M sucrose). The embryos are then placed in BM solution for 5 minutes prior to transfer (Gibbons et al., 2011).

The stage of the embryo is very important. In sheep, morulas and blastocysts have similar survival rates while day 2 embryos have lower survival rate after vitrification (Santos Neto et al., 2015). In goats, blastocysts seem to tolerate vitrification better than morulae and hatched blastocysts (Al Yacoub et al., 2010).

Factors affecting pregnancy rate of frozen-thawed embryos

Several factors may impact the success of embryo cryopreservation and need to be further evaluated. These include method of production of embryos (in vivo vs in vitro), stage of embryo development, breed, age and diet of the donor. Vitrification protocols have been hindered by the toxicity of cryoprotectants. Research efforts have been centered on testing new, less toxic cryoprotectants. Pregnancy rates from vitrified embryos have improved to reach levels that are very comparable to transfer of fresh embryos.

Pregnancy rates following transfer of cryopreserved embryos vary between 38-73% for slow freezing and between 52-79% for vitrification and are not significantly different from those obtained with fresh embryos (50 to 90%) (Baril et al., 2001; Guignot et al., 2006; Hong et al., 2007; Green et al., 2009; Gibbons et al., 2011). There is however an interaction between method of freezing and cryoprotectant used and some vitrification techniques may produce lower pregnancy rates than conventional slow freezing (Varago et al., 2014). In sheep, direct transfer of vitrified embryos resulted in higher pregnancy rates than for embryos frozen by the slow method (Green et al., 2009).

Several factors may affect results of embryo cryopreservation. In vitro produced embryos have lower survival rates than in vivo produced embryos (Zhu et al., 2001; Martinez et al., 2006; Bhat et al., 2015). Pregnancy and birth rates achieved with in vitro produced cryopreserved embryos is also affected by method of culture during production (Mara et al., 2015). The poor survival of in vitro produced embryos may be due to epigenetic alterations (Nieddu et al., 2015; Romão et al., 2015). Also, in goats more advanced stages of development (expanded and hatched blastocysts) survive cryopreservation better than morulas (Gibbons et al., 2011). The effect of breed on embryo ability to withstand cryopreservation merits further studies (Fair et al., 2006). Some the breed effect may be due to differences in lipid content of oocytes and embryos (Romão et al., 2016).

CONCLUSION

Sperm and embryo cryopreservation are established techniques in small ruminants. Incorporation of these biotechnologies in a strategy for the preservation of genetic diversity of small ruminants and propagation of elite sires and dams is a reality. Improvements in sperm preservation through better extenders and reduction of oxidative stress may render the need for laparoscopic artificial insemination in sheep absolute and expand the use of the technique in the field. The development of new more objective methods for evaluation of cryopreserved semen allowed a better understanding of factors affecting post-thaw semen quality. Embryo preservation by vitrification produces similar results as conventional slow methods and reduces the cost of investment in specialized equipment. Further research is needed to refine vitrification techniques for use in the field for direct transfer.

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