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Study contribution
The most used component in mammalian semen cryopreservation extenders is egg yolk. Egg yolk acts as a complex biological compound that contains proteins, vitamins, phospholipids, glucose, and antioxidants potentially beneficial for sperm. However, due to its animal origin, the use of the egg yolk in the extender represents a risk of bacterial contamination and transmission of exotic diseases. This study evaluates the effect of three different extenders: animal protein [Tris egg yolk (Triladyl®)] and animal protein-free extenders (OPTIXcell® and Triladyl®) on ram semen cryopreservation of two tropical hair breeds. The result demonstrated that OPTIXcell® and Triladyl® proved to be the best extenders to cryopreserve Blackbelly and Pelibuey ram semen. However, OPTIXcell® is an animal protein-free extender that decreases the risk of bacterial contamination, whereas Triladyl® is composed of animal protein (egg yolk), which may impact the fertilizing capacity of sperm.
Introduction
The germplasm banks allow the storage of biological materials such as tissues, sperm, oocytes, and embryos, among others, of livestock or wild species of high genetic value, threatened or in danger of extinction, to be used in the future through assisted reproduction techniques.1 One of the tools to achieve the exchange of genetic material between subpopulations that are geographically or biologically isolated is through artificial insemination by using cryopreserved semen. However, a factor that influences this technique is the process of cryopreservation and thawing of semen, which affects the fertilizing capacity of sperm,2 decreasing fertility and prolificacy. Semen extenders contain nutrients and cryoprotective ingredients that are added to semen to preserve the viability and fertilizing capacity of the sperm during and after cryopreservation.3 Among the most used components in mammalian semen extenders is egg yolk.4 Egg yolk acts as a complex biological compound that contains proteins, vitamins, phospholipids, glucose and antioxidants that are potentially beneficial for sperm.5 However, due to its animal origin, the use of the egg yolk in the extender represents a risk of bacterial contamination6,7 and transmission of exotic diseases such as avian influenza.8 In addition, it has endotoxins capable of damaging viability9 and steroid hormones that reduce sperm motility.10 For this reason, the World Organization for Animal Health (WOAH), recommended a sanitary code for terrestrial animals in 2003. They stated that all product of animal origin that is used in the processing of semen must be free of any biological risk.11,12 Likewise, the egg yolk contains granular material that resembles the size and shape of sperm, which makes sperm analysis difficult using computer-assisted semen analysis system13,14 and it can also interfere with microscopic observations or biochemical assays.15 Considering these disadvantages, it was proposed the use of extenders free of animal protein such as soy lecithin that acts in a similar way to the lecithins of the egg yolk16 and liposomes, which are chemically defined molecules that can transfer lipids and cholesterol to the plasma membrane of the sperm, through phosphatidylcholine by changing the transition of the lipid phase of the sperm, making them less sensitive to cooling,17 helping to counteract the damage caused to cells that have been subjected to thermal shocks.18 Therefore, this study aimed to evaluate the effects of two animal protein-free extenders on the cryopreservation of Blackbelly and Pelibuey ram semen.
Materials and methods
Ethical statement
The animal care procedures and research protocols followed the Committee of Ethics on Animal Handling guidelines of the Official Mexican Standard (NOM-027-ZOO-1995), Zoosanitary Process of Domestic Animal Semen.
Place of study
This work was conducted within the autumn (September to October, 2019) at the Instituto Nacional de Investigaciones Forestales, Agrícolas y Pecuarias (INIFAP), by its acronym in Spanish), located in the Mocochá experimental field at kilometer 25 of the old highway Mérida-Motul, located at 21 ° 05 ‘ 18 ‘’ north latitude and 89 ° 27 ‘ 12 ‘’ west longitude, at an altitude of 9 m above sea level. The predominant climate in the region is warm sub-humid (Awo) with a total rainfall of 900 mm and an average annual temperature of 26.5 °C.19
Animals
A total of 8 rams (4 Blackbelly and 4 Pelibuey), with an age of 2 ± 0.5 years and with an average live weight of 43 kg ± 2.95 were used in this study.
Semen collection
A total of 64 ejaculates (8 ejaculates / ram) were obtained per artificial vagina. Ejaculates meeting the following criteria were evaluated: volume > 0.5 mL, motility > 70 %, and sperm concentration > 3000 × 106 sperm/mL.
Sperm concentration
It was determined with the Bürker® chamber after a dilution of 1:200 (995 μL of distilled water + 5 μL of semen).20
Dilution
The ejaculates collected were mixed (pooled), split and diluted to a final concentration of 400 × 106 sperm/mL; with the following extenders: AndroMed® (Minitube, Tiefenbach, Germany) based on soy lecithin; OPTIXcell® (IMV Technologies, L’Aîgle, France) based on liposomes and Triladyl® (Minitube, Tiefenbach, Germany) complemented with 20 % of egg yolk. Subsequently, the diluted samples were packed in 0.25 mL straws (Minitube, Tiefenbach, Germany) for freezing.
Freezing of semen
At the end of the packaging, the straws were stored for 4 h at 5 °C. Subsequently, they will be placed at 4 cm from the surface of the liquid nitrogen (LN2), for 10 minutes, and then immersed in LN2 and stored in cryogenic tanks until evaluation.
Thawing of semen
The thawing procedure was performed by immersing the straws in a water bath at 37 °C for 30 seconds. Subsequently, the samples were evaluated at 0 and 6 h of incubation at 37 °C.
Experimental design
A completely randomized design was used with a factorial arrangement 2 × 3 × 2 with 2 breeds, 3 extenders and 2 times series with 8 repetitions per extender.
Individual and progressive motility
Individual motility was performed by placing 5 μL sample diluted to ~30 × 106/mL of spermatozoa on a Makler® counting chamber (Sefi Medical Instruments, Haifa, Israel), preheated to 37 °C and analyzed using a phase contrast microscope with a 10x objective (UOP, Proiser I + D, Valencia, Spain) the percentage of individual motility on a scale of 0 -- 100 %. And for progressive motility, it was performed by diluting 5 μL of thawed semen in 50 μL extender and it was evaluated on a scale of 0-5, with 5 being the highest degree of motility.
Sperm viability
Sperm viability was evaluated according to the technique described by Nagy et al.,21 with SYBR-14/PI fluorochrome (Live/Dead® kit L-7011, Invitrogen™), adding 1 μL of SYBR-14 (10 µM) and 1 μL IP (12 µM) in 100 μL of sperm sample diluted with saline solution (PBS) and incubated in the dark for 10 minutes at 37 °C. Subsequently, 5 μL of the stained sample was placed on a slide preheated to 37 °C and counted at least 200 sperm with an epifluorescence microscope (LW Scientific i40-ADN, Georgia, US), using the 20x objective and at a wavelength of 488 nm. Those that emitted green fluorescence were considered living cells, while dead cells emitted red fluorescence.
Integrity of the acrosome
The integrity of the acrosome was evaluated using the technique described by Mendoza et al.,22 with the fluorochrome FITC-PSA (100µg/mL, L0770, Sigma-Aldrich™), adding 2 µL of the fluorochrome in 100 μL of sperm sample diluted in PBS and incubated in the dark for 30 minutes at 37 °C. Subsequently, 5 μL of the sample was placed on a slide and were counted 200 sperm with a fluorescence microscope, using the 20x objective and a wavelength of 488 nm. The cells that emitted green fluorescence in the acrosome part were considered damaged.
Mitochondrial activity
Mitochondrial activity was analyzed according to the technique described by Celeghini et al.,23 using the JC-1 fluorochrome (153 µM, Molecular Probes® T-3168, Invitrogen™), mixing 1 μL of the fluorochrome in 100 μL of sperm sample diluted, for 10 minutes at 37 °C. Subsequently, 5 μL of the stained sample was placed on a slide preheated to 37 °C and were counted 200 sperm with a fluorescence microscope, using the 20x objective and with a wavelength of 488 nm. All cells that emitted orange fluorescence in the intermediate part of the flagellum were considered to have mitochondrial activity, while the cells without mitochondrial activity emitted green fluorescence.
The hypo-osmotic swelling test (Host)
The test to determine the plasma membrane integrity of the sperm tail was performed by diluting 5 µL of sperm sample in 50 µL of a hypoosmotic solution composed of 7.35 g/L of sodium citrate and 13.51 g/L of fructose,24 and incubated for 1 h at 37 °C. Subsequently, 5 μL of the sperm sample was placed on a slide and were counted 200 sperm with a phase contrast microscope, using the 20x objective. If the plasma membrane was intact, the sperm had a helical twist of the tail (Positive Host); if in contrast, the membrane was damaged, the spermatozoa did not present a rolled tail (Negative Host).
Data analysis
The distribution of the variables was determined using the Shapiro-Wilk normality test. The variables expressed as percentages (individual motility, viability, acrosomes, mitochondria and PMI) were transformed to arcsine√(variable)/100 and the progressive motility parameter was transformed to √(variable) before its analysis. Subsequently, it was analyzed with a general linear model with procedure (ANOVA) and to determine the statistical differences between extenders, the Tukey test was used at P ≤ 0.05. The results were subjected to a statistical analysis system with the Statistical Analysis System software, SAS.25
Results
The values of the sperm parameters were similar for the OPTIXcell® and Triladyl® extenders, being higher (P < 0.0017) than AndroMed® in TM, PM, viability, and PMI (Table 1). In the Blackbelly ram breed, TM was higher (P < 0.0159) in OPTIXcell® than in AndroMed® (Table 2), while other sperm parameters did not show a significant difference (P > 0.0935) between extenders. On the other hand, in the Pelibuey ram breed, OPTIXcell® and Triladyl® were similar and superior (P < 0.0140) to AndroMed®; in TM, PM, viability, mitochondrial activity and PMI. In the acrosome integrity, Triladyl® was higher (P < 0.0392) than AndroMed®.
Table 1 Effect of extender on frozen-thawed ram semen quality
| Parameters (%) | Extenders | Significance level | ||
|---|---|---|---|---|
| AndroMed® | OPTIXcell® | Triladyl® | ||
| Total motility | 33.66 ± 3.09 b | 43.48 ± 3.43 a | 41.06 ± 2.88 a | ** |
| Progressive motility | 2.69 ± 0.17 b | 3.13 ± 0.15 a | 3.22 ± 0.15 a | ** |
| Viability | 35.87 ± 2.76 b | 43.31 ± 2.26 a | 41.63 ± 2.04 a | ** |
| Acrosome integrity | 39.72 ± 2.97 | 41.16 ± 2.25 | 44.16 ± 2.40 | ns |
| Mitochondrial activity | 43.13 ± 2.74 | 44.56 ± 2.16 | 45.22 ± 1.69 | ns |
| PMI | 29.41 ± 1.34 b | 32.47 ± 1.05 a | 32.69 ± 1.27 a | ** |
Semen quality = (Mean ± SEM).
PMI: Plasma membrane integrity. (a, b) Different literals in the same row show significant differences between extenders. Level of significance between extenders (P < 0.01 **), (P > 0.05 ns).
Table 2 Effect of extender on frozen-thawed semen quality of Blackbelly and Pelibuey ram
| Breed | Parameters (%) | Extenders | Significance level | ||
|---|---|---|---|---|---|
| AndroMed® | OPTIXcell® | Triladyl® | |||
| Blackbelly | Total motility | 36.25 ± 4.82 b | 45.62 ± 2.50 a | 40.62 ± 3.09 ab | ** |
| Progressive motility | 2.56 ± 0.24 | 2.63 ± 0.22 | 2.69 ± 0.20 | ns | |
| Viability | 38.50 ± 3.16 | 44.63 ± 2.49 | 41.31 ± 2.43 | ns | |
| AcrosomeiIntegrity | 37.75 ± 3.70 | 40.13 ± 3.31 | 42.75 ± 3.06 | ns | |
| Mitochondrial activity | 52.25 ± 3.69 | 42.88 ± 3.73 | 45.31 ± 2.49 | ns | |
| PMI | 30.94 ± 2.27 | 32.31 ± 1.77 | 31.81 ± 1.98 | ns | |
| Pelibuey | Total motility | 31.06 ± 3.92 b | 41.25 ± 3.83 a | 41.50 ± 3.19 a | ** |
| Progressive motility | 2.81 ± 0.25 b | 3.63 ± 0.13 a | 3.31 ± 0.20 a | ** | |
| Viability | 31.69 ± 2.03 b | 41.87 ± 2.59 a | 42.88 ± 2.01 a | ** | |
| Acrosome integrity | 37.88 ± 3.10 b | 42.63 ± 2.91 ab | 47.81 ± 2.85 a | * | |
| Mitochondrial activity | 34.00 ± 2.53 b | 46.25 ± 2.23 a | 45.00 ± 2.39 a | ** | |
| PMI | 26.94 ± 1.47 b | 32.13 ± 1.41 a | 32.38 ± 2.08 a | ** | |
Semen quality = (Mean ± SEM).
PMI: Plasma membrane integrity. (a, b) Different literals in the same row showed significant differences between extenders. The level of significance between extenders by breed (P < 0.05 *), (P < 0.01 **), and (P > 0.05 ns)
From 0 to 6 h, the sperm parameters progressively decreased (P < 0.0085) over incubation time similarly in the three extenders and in both breeds (Tables 3 and 4).
Table 3 Effect of extender on frozen-thawed semen quality
| Breed | Sperm parameters (%) | Time (hours) | Extenders | ||
|---|---|---|---|---|---|
| Andromed® | Optixcell® | Triladyl® | |||
| Pelibuey | Total motility | 0 | 41.88 ± 5.17 A | 50.63 ± 5.13 A | 50.63 ± 2.40 A |
| Progressive motility | 3.50 ± 0.27 A | 3.88 ± 0.12 A | 4.00 ± 0.00 A | ||
| Viability | 35.75 ± 2.93 b, A | 47.63 ± 3.90 ab, A | 46.75 ± 2.99 a, A | ||
| Acrosome integrity | 41.63 ± 3.87 b, A | 48.38 ± 3.22 ab, A | 54.88 ± 2.94 a, A | ||
| Mitochondrial activity | 39.63 ± 3.51 A | 48.50 ± 2.99 A | 49.75 ± 2.89 A | ||
| PMI | 31.13 ± 1.78 A | 35.50 ± 1.35 A | 35.75 ± 3.01 A | ||
| Total motility | 6 | 20.25 ± 2.37 b, B | 31.88 ± 3.40 ab, B | 33.12 ± 3.75 a, B | |
| Progressive motility | 2.13 ± 0.23 b, B | 3.38 ± 0.18 a, B | 2.63 ± 0.18 a, B | ||
| Viability | 27.63 ± 2.10 b, A | 36.13 ± 2.05 a, B | 39.00 ± 2.03 a, B | ||
| Acrosome integrity | 34.13 ± 4.71 B | 36.88 ± 4.07 B | 40.75 ± 3.45 B | ||
| Mitochondrial activity | 28.38 ± 2.45 b, B | 44.00 ± 3.29 a, B | 40.25 ± 3.12 a, B | ||
| PMI | 22.75 ± 1.05 b, B | 28.00 ± 1.35 ab, B | 27.75 ± 1.83 a, B | ||
| Significance level | ** | ** | ** | ||
The Pelibuey ram´semen was stored at 37 °C for 6 hours.
PMI: Plasma membrane integrity. (a, b) Different literals in the same row show significant differences between extenders per hour. (A, B) Different literals in the same column show significant differences between sperm parameters over time. The level of significance between groups over time (P < 0.01 **).
Table 4 Effect of extender on frozen-thawed semen quality
| Breed | Sperm parameters (%) | Time (hours) | Extenders | ||
|---|---|---|---|---|---|
| Andromed® | Optixcell® | Triladyl® | |||
| Blackbelly | Total motility | 0 | 50.63 ± 4.47 A | 49.37 ± 1.75 A | 45.62 ± 2.20 A |
| Progressive motility | 3.13 ± 0.12 A | 3.00 ± 0.27 A | 3.13 ± 0.12 A | ||
| Viability | 50.50 ± 2.92 A | 52.50 ± 2.50 A | 48.62 ± 2.42 A | ||
| Acrosome integrity | 53.12 ± 4.79 A | 48.63 ± 3.56 A | 49.38 ± 3.97 A | ||
| Mitochondrial activity | 60.50 ± 4.02 A | 50.88 ± 4.57 A | 51.75 ± 2.50 A | ||
| PMI | 36.25 ± 2.74 A | 36.38 ± 1.58 A | 35.75 ± 2.43 A | ||
| Total motility | 6 | 21.87 ± 4.52 B | 27.87 ± 1.32 B | 24.50 ± 2.74 B | |
| Progressive motility | 2.00 ± 2.38 B | 2.25 ± 0.31 B | 2.25 ± 0.31 B | ||
| Viability | 31.62 ± 3.42 B | 38.12 ± 2.71 B | 32.12 ± 2.29 B | ||
| Acrosome integrity | 30.00 ± 4.70 B | 32.00 ± 3.69 B | 31.62 ± 3.45 B | ||
| Mitochondrial activity | 44.00 ± 4.78 B | 34.88 ± 4.52 B | 39.12 ± 2.90 B | ||
| PMI | 25.87 ± 2.56 B | 28.62 ± 2.48 B | 28.25 ± 2.55 B | ||
| Significance level | ** | ** | ** | ||
The Blackbelly ram´semen was stored at 37 °C for 6 hours (Mean ± SEM).
PMI: Plasma membrane integrity. (A, B) Different literals in the same column show significant differences between sperm parameters over time. The level of significance between groups over time (P < 0.01 **).
Discussion
In vivo tests were excluded from this study. In this sense, different studies are related to post-thawing semen quality where this type of test is not included, such as in the ram,26,27 collared peccary, 28 horses,29 goat,30 among others. Moreover, in vitro fertility does not depend solely on the fertilizing capacity of the sperm, but also on the capacity of the oocyte to be fertilized.31,32 On the other hand, the in vivo test depends largely on the fertility of the female, which is affected by a wide range of factors, such as farm, year, season, artificial insemination technique, and technician,33 heat stress,34-36 diet,37 health status, as well as parity, lambing interval, body condition score, genetic traits, full functionality of reproductive organs, herd management, female prolificacy, nutritional management before and after artificial insemination, the type of estrus (natural or hormonally manipulated), and the site of deposition of the semen and climate factors.38,39
On the other hand, the sperm parameters evaluated in this study were selected due to their predictive capacity in fertility. For instance, several studies have indicated that sperm viability (live/dead) is related to sheep fertility40 and bull fertility41-44 after artificial insemination. Christensen et al.45 and Kumaresan et al.46 reported that the most precise estimation of a bull’s nonreturn rate at 56 days was achieved through the assessment of sperm viability in neat semen and post-thaw sperm viability. Likewise, it has been shown a that a reduced sperm viability is correlated with impaired male fertility.47 The assessment of sperm motility in vitro, it is essential to understand factors which alter motility.48
Poor sperm motility has been associated with low rates of fertilization success in many species.49,50-52 Mass motility, in particular, is useful in ram semen motility assessment due to its correlation to in vivo sperm fertilizing capacity.53 In addition, it has been shown that sperm motility is a convincing indicator of fertility in boar54 and human.55 Mitochondrial activity is related to sperm motility and has been associated to the ability of spermatozoa to fertilize oocytes.56-58 The hypoosmotic swelling test (HOST) has proved to be a good tool for evaluating the plasma membrane integrity of the sperm tail of various domestic animals.
This method contributes to conventional sperm quality tests in predicting the success of in vitro fertilization.59 Oliveira et al.60 reported that the sire that presented numerically lower pregnancy after AI also presented a lower percentage of sperm with a swelling tail. The presence of an acrosome (the acrosomal status) and the ability of the spermatozoa to release the acrosomal content (the acrosomal responsiveness) and expose zona pellucida binding sites in response to female factors correlates with the outcomes of in vitro fertilization.61,62 Kumaresan et al.46 reported that acrosome status is highest correlated with bull’s nonreturn rate at 56 days.
The results of this study showed that the effect of extender on sperm parameters upon thawing are similar to those reported by Stewart et al.63 in semen samples from white-tailed deer and in bulls of the Holstein-Friesian breed,64 where the TM and PM of the spermatozoa were higher with the OPTIXcell® extender upon thawing. Likewise, the viability was also similar to the OPTIXcell® extender in frozen-thawed semen samples of buffalo,18 Holstein bull and Red Swiss bull,65 Korean bull,66 and dromedary camel.67 However, the results obtained from viable spermatozoa differed from those reported by Ondřej et al.68 Frozen-thawed seminal samples from the bull were higher with AndroMed® extender than with OPTIXcell®. In the PMI, the results are like those reported by Ansari et al.18 in bull semen samples frozen with the OPTIXcell® extender, while in the dromedary camel semen, the Triladyl® extender showed a higher percentage of sperm with the membrane integrated upon thawing. The other parameters did not show differences between extenders, being like that reported in frozen-thawed samples of buffalo,18 in Ghezel 69 and Dorper ram,70 in bull65 and rhinoceros.71
In the Blackbelly breed, the results are like those obtained in buffalo semen samples,17 in Balinese bull, 72 in Korean bull,66 and in Bos indicus bull,73 which reported a higher TM in frozen spermatozoa with OPTIXcell® extender. However, Gomes-Alves et al.74 reported that in Brown bear, TM was higher in seminal samples frozen-thawed with the Tris extender complemented with egg yolk than the AndroMed® extender. The sperm parameters PM, viability, acrosome integrity, mitochondrial activity and PMI are similar to those reported by Fleisch et al.75 in frozen-thawed bull semen samples. In the Pelibuey breed, the results are similar to those reported in semen from the Holstein bull and the Swiss red bull,66 where TM, PM, mitochondrial activity, and PMI had higher percentages with the OPTIXcell® extender. However, in acrosome integrity differs from that reported by Singh et al.17 in Bos indicus bull, no differences were found with the extenders AndroMed®, OPTIXcell®, and Triladyl®. In contrast to these results, Souza et al.76 showed that Tris extender improves acrosome integrity more than the Optixcell® extender in frozen/thawed semen sample of Santa Inês ram. These differences between results may be associated with the species and breed of the animal, as well as between individuals within a species.77 This may be attributed, in part, to the lipid composition78,79 and the polyunsaturated fatty acids (PUFAs) content present in the sperm membranes,80 which has an important role in the functionality of the sperm cell, by providing fluidity and permeability, which are related to an increase in cryoresistance.
The sperm parameters evaluated at 0 h and at 6 h of incubation are similar to those found in previous studies with thawed semen samples. TM, PM, acrosome integrity, mitochondrial activity and PMI decreased progressively over time similarly with the extenders AndroMed®, OPTIXcell® and Tris complemented with egg yolk in buffalo semen samples; 0 to 2 hours.81 On one hand, they are similar to those reported by Wojtusik et al.71 in samples of thawed rhinoceros semen; from 0 to 1 h and from a Balinese bull; from 0 to 4 hours73 where TM, PM, viability, acrosome integrity, and PMI decreased progressively over time similarly with extenders OPTIXcell® and Tris complemented with egg yolk. On the other hand, Amal et al.72 mention that the response time of the spermatozoa in incubation will depend on the metabolism of the spermatozoa, as it may depend on the variation of breed and/or species. However, Talini et al.82 quote that the incubation time after thawing does not provide consistent parameters to determine semen quality.
It was observed that the OPTIXcell® extender made from liposomes presented the highest percentage in most of the evaluated sperm variables, compared to the AndroMed® extender made from soy lecithin and the Triladyl® extender complemented with animal protein (chicken egg yolk). The difference between extender (animal protein-free) may be due to the percentage of soy lecithin added to the extender; since it has been proven that high concentrations may be toxic and lower concentrations may be insufficient to protect the plasma membrane from sperm during its cryopreservation.83 On the other hand, Salmin et al.84 mention that the enzyme phospholipase A, that found in the seminal plasma of ram semen can tolerate the levels of soy lecithin, so it does not catalyze the levels of soy lecithin into fatty acids and lysolecithins, causing coagulation in the extender and toxicity in the sperm. Likewise, Ansari et al.18 mentioned that liposomes act by adhering to the sperm, exchanging cholesterol and phospholipids, replacing the lipoproteins in seminal plasma. One or more biological layers of concentric lipids, which help encapsulate molecules85 capable of rehydrating sperm,86,87 counteract the deterioration of phospholipids and the loss of cholesterol88 caused by thermal shocks in freezing-thawing,18 especially in ruminants,64 helping improve the percentage of sperm fertility.
Conclusions
In conclusion, the extenders OPTIXcell® and Triladyl® are the ones that best cryo-preserve Blackbelly and Pelibuey ovine semen. However, OPTIXcell® extender, being free of animal protein in its preparation, reduces the risk of bacterial contamination; unlike Triladyl® extender, which, as it is composed of animal protein (egg yolk), can affect the fertilizing capacity of the sperm.
