Effect of Extender and Freezing Rate on Quality Parameters and In Vitro Fertilization Capacity of Alpaca Spermatozoa Recovered from Cauda Epididymis

Abstract

In alpacas, improvement of reproductive efficiency of male camelids is limited by the small testicular size, low spermatozoa production, and low quality of semen. In this study we aim to evaluate the effect of two extenders and two freezing rates on post-thaw quality of sperm recovered from alpaca epididymis with two methods (flushing and mincing), and to evaluate the in vitro fertilization (IVF) capacity of frozen sperm selected with two different selection methods (washing and swim-up). Sperm samples were processed with Tris–egg yolk or Bioxcell^® extenders and frozen with slow freezing and fast freezing. The oocytes were coincubated with spermatozoa for 72 hours, and cleavage rates were recorded afterward. The results indicated that the recovery method did not influence sperm quality (∼70%). However, total sperm recovery was significantly lower for the flushing method than the mincing method. The sperm quality was influenced by the freezing extender (23.3% vs. 33.2%) and freezing rate (20.9% vs. 35.7%). When comparing different methods of sperm selection for IVF, no differences were observed on cleavage rate except for the fact that the concentration of sperm from swim-up method (20.6%) was significantly lower than the one obtained from the washing method (78.7%). The recovery technique of sperm does not affect sperm quality and the method of fast freezing was shown to be the most effective for cryopreservation of alpaca sperm.

Introduction

Cryopreservation of spermatozoa is an important assisted reproductive technology (ART) that has been investigated in various mammalian species.^1–5 However, successful cryopreservation of South American camelids sperm has had limited progress (∼25% sperm motility post-thawing),^2,5–7 hindering the commercial application of artificial insemination in alpacas.

Camelids have unique anatomical, behavioral, and physiological reproductive characteristics.⁸ Males produce an ejaculate of a low volume, low concentration, and high viscosity.⁹ Also, semen is deposited directly into the uterus. Because of these reasons, semen collection and management techniques are not as well developed as for other domestic animals.¹⁰ In alpacas, sperm is routinely collected as seminal ejaculate with an artificial vagina or, less frequently, by electrical stimulation.¹¹

The recovery, cryopreservation, and fertilizing ability of epididymal sperm have been reported for several domestic species including stallions,¹² boars,¹³ bucks,¹⁴ dogs,^15,16 buffalos,¹⁷ bulls,^18,19 and cats.²⁰ In addition, collection of epididymal sperm may be an option in the case of sudden death of a sire or castration.

Thus, epididymal sperm can easily be used in camelids as a sperm source and model for management, cryopreservation research,²¹ and genetic preservation.⁸ Nonetheless some other factors, influencing sperm cryopreservation, must be considered such as freezing and thawing rates, and also the chosen cryoprotectant and its concentration.²²

Under the in vitro system, there are other considerations to select spermatozoa with good quality to increase the probability of successful fertilization, viable embryos, and healthy offspring. Therefore, several in vitro techniques have been developed to select spermatozoa for their use in ART.²³ The sperm selection techniques evolved from a simple spermatozoa washing to separation techniques based on principles such as migration (swim-up behavior), filtration, or density gradient centrifugation.²⁴ The most simple and cheapest techniques are both the washing (washed twice by centrifugation) and conventional swim-up procedure. Likewise, despite that the first embryos produced by in vitro fertilization (IVF) were reported by Del Campo et al. (1994), in South American camelids, there are few reports on IVF using frozen spermatozoa.^25,26 A combination between intracytoplasmic sperm injection (ICSI), IVF, and cryopreserved sperm might be successfully used for IVF programs that could maximize the efficiency of embryo production. In this study we aim to evaluate the effect of two extenders and two freezing rates on post-thaw quality of sperm recovered from alpaca epididymis with two methods (flushing and mincing), and to evaluate frozen sperm's ability to perform IVF after selection with two different methods (washing and swim-up).

Materials and Methods

Experiment I: comparison of extenders and freezing rates on post-thaw quality in sperm recovered from alpaca epididymis

Sperm recovery

Testicles of adult alpacas (older than 3 years) were collected under hygienic conditions from an alpaca slaughterhouse. These were maintained for 3 hours at a temperature ranging between 25°C and 30°C using isothermal boxes while being transported to the Laboratory of National University of Huancavelica, Peru, located at 3680 m above sea level.

Characteristics of testicular weight (TW), length (TL), and circumference (TC) were determined at the laboratory. Sperm was recovered from the cauda epididymis of a male alpaca randomly selected using either the mincing or flushing method and was processed using two extenders with different compositions. The flushing technique was performed as described by Martinez-Pastor et al.²⁷ Cauda epididymis and ductus deferent were isolated. Sperm cells were flushed with a syringe loaded with ∼1 mL of warm extender in the retrograde direction. Mincing technique was performed as described by Cary et al.²⁸ Cauda epididymis was incised repeatedly with a scalpel in a Petri dish, smashed, and washed in ∼1 mL of warm extender, and tissue debris was discarded. Sperm quality (motility, viability, and membrane integrity) and quantity (concentration) were evaluated 30 minutes after recovery.

Extender and freezing procedure

After recovery, to each sample 2 mL Tris–egg yolk extender or commercial extender (Bioxcell^®) was added. Tris–egg yolk extender (extender 1) was prepared according to the instructions of Evans and Maxwell.²⁹ This freezing extender contains 20% egg yolk and 6% glycerol. Bioxcell (extender 2) was also prepared according to the manufacturer's instructions (IMV, France). After preparing the 3 mL extender (1 and 2) dilution, samples were refrigerated for 2.5 hours at 4°C.

Samples from 19 alpacas were frozen with either slow-freezing or fast-freezing technique.

Slow freezing: Diluted samples were packed in 0.25 mL French plastic straws, placed horizontally in a conditioned grid in an insulated box (16 × 26 × 13 cm) 6 cm above liquid nitrogen (at −10°C, approximately), being exposed to its vapor for 10 minutes, producing a freezing rate of 10°C–15°C/min until achieving −100°C (approximately) to later be submerged into liquid nitrogen. This temperature was verified with cryogenic thermocouples. This technique was described by Salamon and Maxwell,³⁰ Di Santo et al.,³¹ and Thachil and Jewet.³²

Fast freezing: Before freezing, a solid block of dry ice was carved with multiple holes of 0.25 mL capacity. Then, diluted samples were dropped into the dry ice (−79°C) holes and held from 30 to 45 seconds, producing a freezing rate of 110–150°C/min, to later submerge the pellets into liquid nitrogen. This technique was described by Awad and Graham.³³

Sperm analysis

Sperm motility was assessed using a light microscope at × 100 with a previously warmed sample in a heat plate. Viability was evaluated by eosin–nigrosin staining.²⁹ For each smear, 10 μL of diluted sperm was mixed with 20 μL of eosin–nigrosin stain and allowed to air dry for 2 minutes and examined under a light microscope at ×400. Sperm that did not take up the eosin–nigrosin stain was considered alive, whereas those that partially or fully took up the stain were considered dead.³⁴ Functional integrity of the sperm membrane was evaluated with a hypoosmotic swelling (HOS) test. Twenty microliters of semen was added to 80 μL hypoosmotic solution at ∼150 mOsm/kg and incubated for 30 minutes at 37°C. Two hundred spermatozoa per slide were counted under a light microscope at × 400. Spermatozoa that swelled in response to the HOS solution were considered as functional sperm. Sperm concentration was determined using a Neubauer counting chamber (dilution 1:50).

Experiment II: comparison of sperm selection methods on IVF capacity

Oocyte collection and in vitro maturation

Ovaries (n = 335) were collected from alpacas from a local slaughterhouse following the standard procedure described by Ruiz et al.³⁵ These were maintained for 3 hours at a temperature ranging between 25°C and 30°C using isothermal boxes. Cumulus oocyte complexes (COCs) were aspirated from follicles (3–6 mm diameter) using a 21G needle attached to a 10 mL disposable syringe, then were identified and classified under a stereo microscope with ×20 to × 40. Good quality COCs with two layers of compact cumulus cells and homogenous cytoplasm were selected for in vitro maturation.

Oocytes were randomly divided into groups of 10 in 50 μL drops of maturation media under oil in culture dishes. In vitro maturation conditions were similar to those used in bovines: oocytes were matured in vitro using TCM-199 medium with 2.2 g/L NaHCO₃ and 5.94 g/L HEPES supplemented with 10% fetal calf serum (Sigma F-9423), 11 μg/mL sodium pyruvate, 0.02 IU/mL follicle-stimulating hormone, 1 mg/mL estradiol-17β, and 50 μg/mL gentamycin, for 28 hours at 38.5°C and 5% CO₂ in air with 100% humidity, using the procedure described by Ruiz et al.³⁵

Sperm selection and IVF

Both the washing and fertilization media were formulated as described by Parrish et al.³⁶ Fertilization media did not contain any capacitating agent, as described by Conde et al.³⁷ In addition, the straws used for IVF corresponded to the group of Tris–egg yolk–glycerol with slow-freezing method.

Straws (0.25 cc) were thawed in a water bath at 37°C and kept in there from 20 to 30 seconds. Sperm selection by the swim-up method was performed by centrifugation at 503g for 5 minutes with washing media eliminating the supernatant. Sperm was placed in an inclined tube with fertilization medium for 60 minutes and incubated at 5% CO₂ in air at 38.5°C, after which 100 μL were recovered from the surface for use in IVF. The washing method³⁸ consisted of washing the sperm twice in washing media by centrifugation at 503g for 5 and 3 minutes, respectively, and recovering 50 μL from the bottom of the tube for usage in IVF. The final spermatozoa concentration for oocyte fertilization was 5.0 × 10^5/mL. For IVF, 10 IVM oocytes were washed with IVF media and shifted to fertilization drops (45 μL) under oil, made in separate Petri dishes for separate IVF. Oocytes were inseminated with 5000 spermatozoa:1 oocyte (5 × 10⁵ sperm mL⁻¹), then were coincubated with spermatozoa for 72 hours at 38.5°C, and cleavage rates were recorded afterward. The cleavage rate equals the number of embryos with ≥2 cells divided by the number of oocytes.

Statistical analysis

The results were expressed as mean ± standard deviation and statistical significance was considered at p < 0.05. Data were analyzed using the IBM SPSS Statistics program for Windows, Version 19.0 (IBM, Armonk, NY) and through both the Shapiro–Wilk test and Levene test to check the assumption of normality and homogeneity of variances.

The mean values of testis (TW, TL, and TC) and sperm parameters (total sperm recovery, motility, viability, and plasma membrane integrity) were compared using one-way analysis of variance (ANOVA). The sperm motility, sperm viability, and plasma membrane integrity post-thawing were compared using factorial ANOVA for 2 × 2 independent groups: two extenders (Bioxcell and Tris–egg yolk–glycerol) and two freezing rates (slow freezing and fast freezing). Cleavage rates (≥2 cells) on IVF were compared using one-way ANOVA test for the washing and swim-up methods.

Results

Experiment I: comparison of extenders and freezing rates on post-thaw quality in sperm recovered from alpaca epididymis

The recovery method did not influence sperm parameters such as motility, viability, and plasma membrane integrity (Table 1). However, total sperm recovery (p = 0.005) was significantly lower for the flushing method (25.2 × 10⁶, range 5–62.5 × 10⁶ spermatozoa) than for the mincing method (51.7 × 10⁶, range 7.5–117.5 × 10⁶). Testicular parameters measured individually (left or right testicles) showed no difference (p > 0.05), consequently, they did not have any effect on the recovery techniques. In addition, sperm samples recovered from alpaca epididymis with flushing and mincing methods showed motility (∼70%) and viability (∼70%).

Table 1.

Testicular and Sperm Parameters from Cauda Epididymal in Alpaca (Mean ± Standard Deviation)

	Sperm recovery technique
Parameters	Mincing	Flushing	p
Male	19	19	—
Testicular parameters
Testicular weight, g	12.6 ± 4.0	12.3 ± 3.8	0.813
Testicular length, mm	35.7 ± 4.6	35.1 ± 4.8	0.681
Testicular circumference, mm	72.5 ± 8.2	72.1 ± 7.6	0.894
Sperm parameters
Total sperm recovery ( × 10⁶)	51.7 ± 34.7	25.2 ± 17.6	0.005
Motility, %	69.7 ± 12.1	70.3 ± 10.5	0.887
Viability, %	75.7 ± 9.9	71.7 ± 10.7	0.251
Membrane integrity, %	35.6 ± 16.4	40.0 ± 13.1	0.638

Sperm motility and viability were significantly influenced by the freezing extender and freezing rate (Table 2), although there were no significant differences for the percentage of membrane integrity. In addition, factorial ANOVA showed no significant interactions between the freezing extender and freezing rate.

Table 2.

Post-Thaw Sperm Parameters from Cauda Epididymis in Alpaca (Mean ± Standard Deviation)

Parameters	Technique 1	Technique 2	—
Samples	38	38	—
Prefreezing motility, %	68.8 ± 9.6	72.9 ± 11.2	—
Comparison of two different freezing extenders
Sperm parameters	Tris–egg yolk–glycerol	Bioxcell^® extender	p
Motility, %	23.3 ± 14.2	33.2 ± 16.8	0.003
Viability, %	32.0 ± 14.2	38.4 ± 14.4	0.047
Membrane integrity, %	18.6 ± 7.9	14.9 ± 10.5	0.089
Comparison of two different freezing rates
Sperm parameters	Slow freezing	Fast freezing	p
Motility, %	20.9 ± 12.3	35.7 ± 16.5	0.0001
Viability, %	30.4 ± 14.1	40.0 ± 13.6	0.003
Membrane integrity, %	16.5 ± 9.3	17.0 ± 9.6	0.824

Experiment II: comparison of sperm selection methods on IVF capacity

The initial progressive motility (25.0% ± 5.9%), viability (32.0% ± 5.6%), and concentration (66.6 ± 6.1 × 10⁶ mL⁻¹) of sperm were analyzed in the samples after thawing. After sperm selection, the concentration of spermatozoa recovered from the swim-up method (3.4 ± 2.7 × 10⁶ sperm; 20.6% of initial) was significantly lower than that obtained from the washing method (13.0 ± 7.1 × 10⁶ sperm; 78.7% of initial) (p < 0.05).

A total of 828 COCs of good quality were collected from 335 ovaries (an average of 2.5 COCs per ovary), and at 72 hours after sperm–oocyte incubation we identified 300 zygotes that developed from 2 to 8 cells (cleavage). There was no effect of sperm selection method (p = 0.943) on alpaca fertilization rate (Table 3).

Table 3.

Effect of Swim-Up and Washing Method for Selection of Spermatozoa on Cleavage Development In Vitro

Selection method	Repeated	No. of oocytes examined	Cleavage (72 hours)	Fertilization rate (%)
Swim-up	12	413	147	36.6 ± 16.2
Washing	12	415	153	36.0 ± 20.7

Discussion

In this study, the testicle measurements (weight, ∼12 g; length, ∼35 mm; circumference, ∼72 mm) were similar to a report in adult male alpaca of 17.2 g (TW).³⁹ Thus, TC is often used because it is easier to measure and displays a high correlation with body weight and reproductive capacity (libido), particularly with sperm production,⁴⁰ although TC is difficult to measure in alpacas because of the small size of the testicles and their anatomical location (very close to the abdomen). Abraham et al.⁴¹ concluded that TL is better to predict sperm production and suggested that a combination of this parameter with body condition scores could be a tool for decision making in the selection of potential alpaca male sires for animal husbandry under Swedish conditions.

In South American camelids, the collection of semen presents many difficulties because of the nature of their copulatory behavior and slow process (dribbling) of ejaculation. The main techniques for semen collection used in practice for these species are the artificial vagina, electroejaculation, or postcoital aspiration from a female.⁴² In addition, the use of epididymal sperm acts as a potential source of gametes. Recovery and cryopreservation of sperm from the epididymides of slaughtered animals is an alternative tool to recover sperm, especially in wild species and in situations where traditional collection is difficult because of the lack of expertise and/or facilities.¹⁹

Although sperm concentration is highly variable and is affected by age, method of collection, and ejaculated rank,⁴³ the amount of sperm recovered by the mincing technique was significantly superior (51.7 × 10⁶ spermatozoa) than the amount recovered by the flushing technique (25.2 × 10⁶ spermatozoa). Several researchers have compared these recovery methods (flushing and mincing) in other species such as stallions,²⁸ red deer,²⁷ goats,⁴⁴ bulls,¹⁹ and dogs.¹⁵ The flushing technique is recommended for postmortem recovery of sperm from the cauda epididymis. In addition, the results indicate that the recovery method of alpaca sperm from the cauda epididymides did not affect its sperm quality (Table 1). Post-thaw motility has rarely exceeded 40% for camelid semen, although there have been reports of >45% for alpaca,⁴⁵ 60% for bactrian camel sperm,⁴⁶ and 50% for dromedary camel sperm.⁴⁷ In South American camelids, information is limited and processes are very variable and not repeatable. Some of the publications have reported poor results in alpaca^2,21,40 (<20%) and llama⁵ (<25%). In this work, cryopreserved sperm showed sperm motility and viability improved using the method of fast freezing (35.7% and 40.0%, respectively) and Bioxcell extender (33.2% and 38.4%, respectively). However, freezing spermatozoa in straws provides a more uniform freeze rate because the entire sample surface area is exposed to nitrogen vapor,⁴⁸ compared with freezing spermatozoa in pellets, because one side of the pellet is in contact with a cold surface, whereas the other side is not. However, Awad and Graham³³ reported that ram spermatozoa survived cryopreservation more efficiently when frozen in pellet form, whereas bull spermatozoa survived more efficiently when frozen in straws. Salamon and Maxwell³⁰ reported that freezing and thawing spermatozoa cause ultrastructural, biochemical, and functional damage to a significant proportion of the cells. These problems are minimized when alpaca spermatozoa are frozen in pellets (fast freezing) rather than frozen in straws. This favorable effect of a rapid freezing rate was observed in a higher number of replicates when freezing sperm from llama.⁵ In addition, Morton et al.²¹ reported motility percentages <27% from alpaca epididymal sperm frozen in pellets using dry ice. This difference can be influenced by the dosage volume.^21,49 In general, sperm damage during freezing has been attributed to several factors including cold shock, freezing injury, oxidative stress, alterations in membrane compositions, chemical toxicity of cryoprotective agents, and osmotic stress.⁵⁰

Several of the commonly used extenders for ruminant semen preservation have been applied to semen from alpaca and llama. Vaughan et al.⁴⁹ worked with five extenders (Sheep Red, Green Camel, Triladyl, Biladyl, and Andromed) showing motility results <30% after 24 hours. However, a general evaluation of these findings suggests that Tris-buffered extenders may be more suitable than others and the use of a 7% glycerol concentration was superior in terms of viability and motility.⁵¹ In addition, other studies have affirmed that Tris-extender may be promising for further fertility trials of cryopreserved semen, but centrifugation of prediluted semen would probably be necessary to get a minimum amount of sperm into the straw.⁵ Also, the addition of an antioxidant can improve the individual post-thawing motility to 42.3%.⁵² In llamas, post-thaw motility was greater (p < 0.05) in lactose–EDTA–egg yolk (LEEY) extender with 7% dimethylformamide (LEEY-DMF) than LEEY extender with 7% glycerol.⁴

In bovines, frozen semen for IVF programs is widely used.⁵³ However, in South American camelids, there are very few works indicating the use of alpaca frozen sperm for IVF.²⁵ Also, it is important to clarify that preparation of maturation, fertilization, and in vitro culture medium in camelids is based on the preparation of medium applied in bovines. Therefore, it remains necessary to find an adequate culture medium and conditions that favor embryo development.²⁶

In mammals, swim-up and Percoll gradient centrifugation methods are mostly used for semen manipulation techniques for IVF systems. However, comparison studies of swim-up and Percoll gradient centrifugation separation methods showed controversial results in different species, such as humans,⁵⁴ bovines,⁵⁵ stallions,⁵⁶ and pigs.⁵⁷ In our study, we used the most simple and cheap methods for sperm selection. The concentration of spermatozoa recovered from the swim-up method (3.4 ± 2.7 × 10⁶ sperm; 20.6% of initial) was significantly lower than those obtained from the washing method (13.0 ± 7.1 × 10⁶ sperm; 78.7% of initial) (p < 0.05). Thus, we can assume that the sperm selected by the washing method is better and can fertilize 3.8 times more oocytes. Similar results in yak were reported by Liu et al.⁵⁸ Sperm separated by the swim-up method showed better motility (76.7% vs. 48.8%), whereas the concentration of recovered spermatozoa was higher with Percoll gradient centrifugation (4.06 × 10⁷/mL; 55.5% of initial vs. 1.48 × 10⁷/mL; 20.2% of initial). In addition, Parrish,⁵³ working with bull, reported that recovery of motile sperm from frozen–thawed semen was 9% ± 1% for swim-up approach but 40% ± 4% for Percoll gradient centrifugation.

In our results, when comparing different methods of sperm selection for IVF, no difference on cleavage rate (36%) was observed. Similar results of cleavage rate were reported in alpaca IVF using frozen sperm by Gamarra et al.²⁵ (27.1% of cleavage) and with fresh sperm by Huanca et al.⁵⁹ (15.4% of cleavage rate) and Perez et al.⁶⁰ (27.5% of cleavage). Ruiz et al.³⁵ reported very interesting rates of cleavage (66%) and blastocyst (20%) using fresh sperm. In some clinical studies, frozen–thawed sperm had lower fertilization ability compared with fresh sperm.⁶¹

Conclusion

The mincing technique is simple and does not affect sperm quality. Also, the method of fast freezing was shown to be the most effective for cryopreservation for alpaca epididymal spermatozoa. However, comparing different sperm selection methods for IVF capacity with frozen sperm collected from alpaca epididymides, no differences freezing of cleavage rate were observed. Hence, swim-up freezing epididymal spermatozoa is an effective and practical method of preserving male alpaca genetic potential.

Authors' Contributions

E.M.S., M.M.G., G.M.M., and M.R.H. conceived and designed the work. E.M.S., M.M.G., M.R.H., G.M.M., J.M.M., and J.R.B. performed the experiments and analyzed the data. E.M.S., V.R.P., and G.M.M. wrote the article. All authors participated in editing the manuscript and approved its final version.

Footnotes

Author Disclosure Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

References

Agca

, Critser

. Cryopreservation of spermatozoa in assisted reproduction. SeminReprod Med, 2002; 20:15–23.

Morton

, Evans

, Maxwell

. Effect of glycerol concentration, Equex STM supplementation and liquid storage prior to freezing on the motility and acrosome integrity of frozen-thawed epididymal alpaca (Vicugnapacos) sperm. Theriogenology, 2010; 74:311–316.

Santiani

, Evangelista

, Valdivia

, Risopatrón

, Sánchez

. Effect of the addition of two superoxide dismutase analogues (Tempo and Tempol) to alpaca semen extender for cryopreservation. Theriogenology, 2013; 79:842–846.

Carretero

, Neild

, Ferrante

, et al. Effect of cryoprotectant and equilibration temperature on cryopreservation of Lama glama spermatozoa. Andrologia, 2015; 47:685–693.

Von Baer

, Hellemann

. Cryopreservation of Llama (Lama glama) Semen. Reprod Domest Anim, 1999; 34:95–96.

Bravo

, Ccallo

, Garnica

. The effect of enzymes on semen viscosity in Llamas and Alpacas. Small Rumin Res, 2000; 38:91–95.

Santiani

, Huanca

, Sapana

, Huanca

, Sepulveda

, Sanchez

. Effects on the quality of frozen-thawed alpaca (Lama pacos) semen using two different cryoprotectants and extenders. Asian J Androl, 2005; 7:303–309.

Abraham

, Verdier

, Båge

, Morrell

. Semen collection methods in alpacas. Vet Rec, 2017; 180:613–614.

Kershaw-Young

, Maxwell

. Advancing artificial insemination in camelids, particularly the alpaca. RIRDC Publication No 11(added by RIRDC) RIRDC Project No R1882. 2011. 99p. ISSN 1440–6845. https://cdn.harper-adams.ac.uk/document/project/150604-Advancing-Artificial-Insemination-in-Alpacas423506.pdf, Accessed January 10, 2018.

10.

Stuart

, Bathgate

. Advancing assisted reproductive technologies in camelids (especially the alpaca) Publication No. 15/067 Project No. PRJ-006417. 2015. p. 36. https://agrifutures.infoservices.com.au/items/15-067, Accessed January 10, 2018.

11.

Leboeuf

, Restall

, Salamon

. Production and storage of goat semen for artificial insemination. Anim Reprod Sci, 2000; 62:113–141.

12.

Papa

, Melo

, Fioratti

, Del'Aqua

Jr. , Zahn

, Alvarenga

. Freezing of stallion epididymal sperm. Anim Reprod Sci, 2008; 107:293–301.

13.

Nagai

, Takahashi

, Masuda

, et al. In-vitro fertilization of pig oocytes by frozen boar spermatozoa. J Reprod Fertil, 1988; 84:585–591.

14.

Blash

, Melican

, Gavin

. Cryopreservation of epididymal sperm obtained at necropsy from goats. Theriogenology, 2000; 54:899–905.

15.

Hori

, Atago

, Kobayashi

, Kawakami

. Influence of different methods of collection from the canine epididymides on post-thaw caudal epididymal sperm quality. J Vet Med Sci, 2015; 77:625–630.

16.

Martins

MIM

, Padilha

, Souza

, Lopes

. Fertilizing capacity of frozen epididymal sperm collected from dogs. Reprod Dom Anim, 2009; 2:342–344.

17.

Herold

, Aurich

, Gerber

. Epididymal sperm from the African buffalo (Synceruscaffer) can be frozen successfully with AndroMed and with Triladyl but the addition of bovine seminal plasma is detrimental. Theriogenology, 2004; 61:715–724.

18.

Chaveiro

, Cerqueira

, Silva

, Franco

, Moreira da Silva

. Evaluation of frozen thawed cauda epididymal sperms and in vitro fertilizing potential of bovine sperm collected from the cauda epididymal. Iran J Vet Res, 2015; 16:188–193.

19.

Turri

, Madeddu

, Gliozzi

, Gandini

, Pizzi

. Influence of recovery methods and extenders on bull epididymal spermatozoa quality. Reprod Domest Anim, 2012; 47:712–717.

20.

Tsutsui

, Wada

, Anzai

, Hori

. Artificial insemination with frozen epididymal sperm in cats. J Vet Med Sci, 2003; 65:397–399.

21.

Morton

, Bathgate

, Evans

, Maxwell

CWM

. Cryopreservation of epididymal alpaca (Vicugnapacos) sperm: A comparison of citrate-, Tris- and lactose-based diluents and pellets and straws. Reprod Fertil Dev, 2007; 19:792–796.

22.

Royere

, Barthelemy

, Hamamah

, Lansac

. Cryopreservation of spermatozoa: A 1996 review. Hum Reprod Update, 1996; 2:553–559.

23.

Morrell

, Rodríguez-Martínez

. Biomimetic techniques for improving sperm quality in animal breeding: A review. The Open Andrology J, 2009; 1:1–9. https://pdfs.semanticscholar.org/4fd9/eee42cdad7d1f2cf2a7d5e1d605f68424bb2.pdf, Accessed January 10, 2018.

24.

Makker

, Agarwal

, Sharma

. Magnetic activated cell sorting (MACS): Utility in assisted reproduction. Indian J Exp Biol, 2008; 46:491–497.

25.

Gamarra

, Huaman

, León

, et al. 157 First in vitro embryoproduction in alpacas (Lama pacos). Reprod Fertil Dev, 2008; 21:177–178.

26.

Trasorras

, Giuliano

, Miragaya

. In vitro production of embryos in South American camelids. Anim Reprod Sci, 2013; 136:187–193.

27.

Martinez-Pastor

, Garcia-Macias

, Alvarez

, et al. Comparison of two methods for obtaining spermatozoa from the cauda epididymis of Iberian red deer. Theriogenology, 2006; 65:471–485.

28.

Cary

, Madill

, Farnsworth

, Hayna

, Duoos

, Fahning

. A comparison of electroejaculation and epididymal sperm collection techniques in stallions. Can Vet J, 2004; 45:35–41.

29.

Evans

, Maxwell

WMC

. Salamon's Artificial Insemination of Sheep and Goats. Australia: Butterworths; 1987: 122–141.

30.

Salamon

, Maxwell

WMC

. Frozen storage of ram semen. 1. Processing, freezing, thawing and fertility after cervical insemination. Anim Reprod Sci, 1995; 37:185–249.

31.

Di Santo

, Tarozzi

, Nadalini

, Borini

. Human sperm cryopreservation: Update on techniques, effect on DNA integrity, and implications for ART. Adv Urol, 2012; 2012:854837.

32.

Thachil

, Jewett

. Preservation techniques for human semen. Fertil Steril, 1981; 35:546–548.

33.

Awad

, Graham

. 2004. A new pellet technique for cryopreserving ram and bull spermatozoa using the cold surface of cattle fat. Anim Reprod Sci, 2004; 84:83–92.

34.

Memon

, Wahid

, Rosnina

, Goh

, Ebrahimi

, Nadia

. Effect of antioxidants on post thaw microscopic, oxidative stress parameter and fertility of Boer goat spermatozoa in Tris egg yolk glycerol extender. Anim Reprod Sci, 2012; 136:55–60.

35.

Ruiz

, Santayana

, Mendoza

, et al. Effect of oocyte maturation time, sperm selection method and oxygen tension on in vitro embryo development in alpacas. Theriogenology, 2017; 95:127–132.

36.

Parrish

, Susko-Parrish

, Winer

, First

. Capacitation of bovine sperm by heparin. Biol Reprod, 1988; 38; 1171–1180.

37.

Conde

, Herrera

, Trasorras

, et al. In vitro production of llama (Lama glama) embryos by IVF and ICSI with fresh semen. Anim Reprod Sci, 2008; 109:298–308.

38.

Chauhan

, Nadir

, Bailey

, et al. Bovine follicular dynamics, oocyte recovery, and development of oocytes microinjected with a green fluorescent protein construct. J Dairy Sci, 1999; 82:918–926.

39.

Bravo

. Chapter15. Reproductive anatomy and physiology in the male. In: Cebra

, Anderson

, Tibary

, Van Saun

, Willard Johnson

(eds). Llama and Alpaca Care: Medicine, Surgery, Reproduction, Nutrition, and Herd Health. Canada: Elsevier, Inc.; 2014:150–161.

40.

Brito

, Silva

, Unanian

, Dode

, Barbosa

, Kastelic

. Sexual development in early and late maturing Bos indicus and Bos indicus x Bos taurus crossbred bulls in Brazil. Theriogenology, 2004; 62:1198–1217.

41.

Abraham

, Puhakka

, Ruete

, et al. Testicular length as an indicator of the onset of sperm production in alpacas under Swedish conditions. Acta Vet Scand, 2016; 58:10.

42.

Pacheco-Curie

. Métodos de colección de semen en camélidos sudamericanos. REDVET. Revista electrónica de Veterinaria 2008. 1695–7504. IX, 040806. www.veterinaria.org/revistas/redvet/n040408/040806.pdf, Accessed January 10, 2018.

43.

Tibary

, Pearson

, Anouassi

. Chapter 16. Applied andrology in camelids. In: Chenoweth

, Lorton

(eds). Animal andrology: Theories and applications. Oxfordshire, UK: CAB International; 2014: 418–449.

44.

Santiago-Moreno

, Astorga

, Luque

, et al. Influence of recovery method and microbial contamination on the response to freezing-thawing in ibex (Capra pyrenaica) epididymal spermatozoa. Cryobiology, 2009; 59:357–362.

45.

Bravo

, Ordonez

, Alarcon

. Processing and freezing of semen of alpacas and llamas. Proceedings 13th International Congress on Animal Reproduction Sydney, Australia, 1996: 2–3.

46.

Zhao

, Huang

, Nie

, Zhang

, Chen

. Effect of different extenders on motility survival and acrosomal integrity of camel spermatozoa frozen in ampoules. J Camel Sci, 2004; 1:31–36.

47.

Malo

, Crichton

, Skidmore

. Optimization of the cryopreservation of dromedary camel semen: Cryoprotectants and their concentration and equilibration times. Cryobiology, 2017; 74:141–147.

48.

Robbins

, Saacke

, Chandler

. Influence of freeze rate, thaw rate and glycerol level on acrosomal retention and survival of bovine spermatozoa frozen in French straws. J Anim Sci, 1976; 42:145–154.

49.

Vaughan

, Galloway

, Hopkins

. Artificial insemination in alpacas (Lama pacos), RIRDC project; no. AAA-1A.: Rural Industries Research and Development Corporation, 2003: 90.

50.

Critser

, Mobraaten

. Cryopreservation of murine spermatozoa. ILAR J, 2000; 41:197–206.

51.

Bravo

, Alarcon

, Baca

, et al. Semen preservation and artificial insemination in domesticated South American camelids. Anim Reprod Sci, 2013; 136:157–163.

52.

Gómez-Quispe

, Guido Pérez

, Machaca

. Addition of the tempol antioxidant in cryopreservation and spermazoa of Alpaca (Vicugna pacos) collected by deviations of the vas deferens. (Spanish). Revista de Investigaciones Veterinarias del Perú, 2016; 27:294–302.

53.

Parrish

. Bovine in vitro fertilization: In vitro oocytematuration and spermcapacitationwithheparin. Theriogenology, 2014; 81:67–73.

54.

Donnelly

, McClure

, Lewis

. Cryopreservation of human semen and prepared sperm: Effects on motility parameters and DNA integrity. Fertil Steril, 2001; 76:892–900.

55.

Cesari

, Kaiser

, Mucci

, et al. Integrated morphophysiological assessment of two methods for sperm selection in bovine embryo production in vitro. Theriogenology, 2006; 66:1185–1193.

56.

Sieme

, Martinsson

, Rauterberg

, et al. Application of techniques for sperm selection in fresh and frozen-thawed stallion semen. Reprod Domest Anim, 2003; 38:134–140.

57.

Matás

, Coy

, Romar

, Marco

, Gadea

, Ruiz

. Effect of sperm preparation method on in vitro fertilization in pigs. Reproduction, 2003; 125:133–141.

58.

Liu

, Cui

, Yu

. Effect of swim-up and percoll treatment on sperm quality and in vitro embryo development in yak. J Integr Agric, 2013; 12:2235–2242.

59.

Huanca

, Condori

, Cainzos

, et al. 341 In vitro maturation and in vitro fertilization of alpaca (Vicugnapacos) oocytes: Effect of time of incubation on nuclear maturation and cleavage. Reprod Fertil Dev, 2009; 22:327–327.

60.

Pérez

, Zevallos

, Pérez

. Comparison of alpaca embryo culture systems. (Spanish). Revista de Investigaciones Altoandinas, 2017; 19:157–164.

61.

Geraci

, Giudice

. Factors which influence sperm ability to fertilize. J Submicrosc Cytol Pathol, 2005; 37:215–222.