Study of Sperm Evaluation Parameters to Estimate Cryopreserved Bovine Semen Fertility

Research Article

Austin J Vet Sci & Anim Husb. 2015;2(1): 1005.

Study of Sperm Evaluation Parameters to Estimate Cryopreserved Bovine Semen Fertility

Morado S1*, Pereyra V1, Breininger E1, Sara R2 and Cetica P1,3

1Institute of Research and Technology in Animal Reproduction, University of Buenos Aires, Argentina

2Institute of Research in Animal Production, University of Buenos Aires, Argentina

3Bovine Reproduction Centre, Argentina

*Corresponding author: Morado S, Institute of Research and Technology in Animal Reproduction, Area of Biochemistry, School of Veterinary Science, University of Buenos Aires, Chorroarin 280, Chair of Biological Chemistry, University of Buenos Aires, Buenos Aires, Argentina

Received: October 13, 2014; Accepted: February 22, 2015; Published: February 24, 2015

Abstract

In cattle production, reproductive biotechnologies such as artificial insemination with Cryopreserved semen samples show the same performance as insemination with fresh semen. The most representative method to evaluate semen fertilizing capability is through the results obtained by in vivo fertility. However, as it is difficult to apply this method in practice, routine laboratory tests can be used to evaluate semen quality. The aim of this work was to evaluate functional spermatic parameters in cryopreserved bovine semen samples from males presenting high and low field fertility rates. Semen samples were obtained from bulls presenting low or high fertility in a program of artificial insemination. Routine and functional parameters were determined in both groups. As regards routine tests, no significant differences were observed in progressive motility and vigor between samples of animals with high and low fertility in vivo, but differences were detected in vitality and acrosome integrity (p<0.05). Within functional tests, no differences were detected in thermo resistance and capacitation induction, but HOS test, acrosomal reaction induction and IVF showed significant differences (p<0.05). These results assert that routine parameters (progressive motility and vigor) have a limited value to predict field fertility. Therefore, the incorporation of some functional tests may offer a better estimation of fertilizing capability.

Keywords: Bull sperm; Semen fertility; Functional parameters; In vitro fertilization

Abbreviations

IVF: In Vitro Fertilization; ICSI: Intracytoplasmic Sperm Injection; HOS test: Hypoosmotic Swelling Test; TALP: Tyrode's Albumin Lactate Pyruvate; BSA: Bovine Serum Albumin; MSOF: Modified Synthetic Oviductal Fluid; TB: Trypan Blue; ANOVA: Analysis of Variances; AI: Artificial Insemination

Introduction

In cattle production, reproductive biotechnologies such as Artificial Insemination (AI) with cryopreserved semen samples show the same performance as insemination with fresh semen. The most representative method to evaluate semen fertilizing capability is through the results obtained by in vivo fertility. However, as it is difficult to apply this method in practice, routine laboratory tests (progressive motility, vigor, vitality and acrosome integrity) can be used to evaluate semen quality, although their usefulness to predict semen fertility is limited mainly due to the complexity of sperm and the fertilization process [1]. Some studies report that In Vitro Fertilization (IVF) developed with frozen semen from bulls of high fertility yielded higher cleavage and blastocyst formation rates [2].

The study of the sperm membrane functional status is of particular importance since an intact and functionally active membrane is required for cell metabolism, capacitation, acrosome reaction, attachment and penetration of the oocyte [3]. The elucidation of these mechanisms is of fundamental importance to resolve cases of infertility and to choose the optimal conditions for the performance of assisted reproductive techniques, such as IVF and Intracytoplasmic Sperm Injection (ICSI) [4,5]. Thus, the assessment of the sperm membrane functional status appears to be a significant marker for the fertilizing capacity of spermatozoa [6]. The Hypoosmotic Swelling (HOS) test was designed to evaluate the function of the sperm membrane [3].

The fertility of a bull has traditionally been evaluated by test inseminations in the field and while this method is considered reliable, it is expensive and time consuming [7]. Consequently it would be a benefit for the cattle industry to have an accurate, simple and efficient in vitro method of predicting the potential fertility of semen, where aspects such as time, cost and practicability are considered [8].

Therefore, the aim of this work was to evaluate routine and functional sperm parameters in cryopreserved bovine semen samples from males presenting high and low field fertility rates.

Materials and Methods

Semen samples were obtained from four bulls (Aberdeen Angus, 15-20 months of age) which belonged to a bovine reproduction center and were being evaluated for their possible inclusion in a controlled program of AI. These bulls presented different performances in preliminary field tests, showing low (below 60% pregnancy rate) or high field fertility rates. The semen pellets from bulls presenting low (1 sample, 5 replicates per sample) or high (3 samples, 5 replicates per sample) were thawed in Tyrode's Albumin Lactate Pyruvate (TALP) medium without calcium or Bovine Serum Albumin (BSA) at 37°C in a 1:2 ratio. After equilibration (10 min), samples were centrifuged at 300 xg for 5 min to separate seminal plasma and freezing extender. The pellet was resuspended in the same medium and centrifuged as described before. Samples were then suspended in a TALP medium containing 2 mm CaCl2 and 6 mg/ml BSA (1.5x107 sperm/ml) to perform the routine and functional tests.

Evaluation of progressive motility and sperm vitality

Progressive motility and vigor were evaluated by light microscopy (x400) with a thermal stage (37°C) three times by the same observer after each treatment (45 min). The percentage of live spermatozoa was determined by the supravital eosin/nigrosin technique. At least 200 spermatozoa were counted in each sample.

Acrosome integrity

Two hundred spermatozoa were evaluated to determine acrosome integrity in live cells with the combined technique of 0.25% trypan blue and differential interference optical contrast.

Thermoresistance test

The capability of spermatozoa to resist incubation at 37°C for 2h was evaluated in each treatment assessing the progressive motility.

HOS test

HOS test was performed by adding 50 μl of sperm suspension to 200 μl fructose-citrate hypoosmotic solutions (50 MSOF) and incubating for 30 minutes. Two hundred spermatozoa were evaluated at the end of incubation to determine the swelling patterns.

Capacitation induction

Sperm suspensions were incubated for 45 min at 38°C under 5% CO2 in humidified air in the presence of heparin (capacitation inducer). Determination of capacitation was made by the chlortetracycline epifluorescence assay using an epifluorescence microscope (Jenamed 2, Carl Zeiss, Jena, Germany) at x400. Two hundred sperm were examined and designated as one of the following: F-pattern (intact sperm), where the fluorescence was detected over the whole region of the sperm head; B-pattern (capacitated sperm), where the fluorescence was detected in the sperm head, except in the post-acrosomal region; and AR-pattern (acrosome reacted sperm), with no head fluorescence. The percentages of the B and AR patterns were obtained by subtracting from the values obtained in the control and treated samples, the ones obtained at zero time, in order to rule out cells destabilized during the freezing-thawing process.

Acrosome reaction induction and evaluation

The ability of the capacitated sperm to undergo acrosome reaction was assessed as follows: samples were incubated with bovine follicular fluid (30% (v/v) for 15 min in the same capacitating conditions). The acrosome reaction was evaluated by a combined technique of differential-interferential contrast microscopy and a supravital stain Trypan Blue (TB). Sperm (200/sample) were assessed at x1000 magnification (Carl Zeiss Jenamed 2 microscope, Jena, Germany). The percentage of live acrosome reacted sperm was evaluated by counting the percentage of live acrosome reacted sperm in each treatment from which the one obtained at zero time was subtracted, in order to rule out cells destabilized during the freezing-thawing process.

In vitro fertilization

IVF was carried out using cryopreserved semen samples from bulls of low and high field fertility rates. Semen was thawed at 37°C in Modified Synthetic Oviductal Fluid (MSOF) [9] with 10 mmol/l theophylline, centrifuged at 500 xg twice for 5 min and then resuspended in fertilization medium to a final concentration of 2 x106 motile spermatozoa/ml. Co-incubation of COCs and spermatozoa was performed in IVF-MSOF medium, consisting of MSOF supplemented with 10 IU/ml heparin and 5 mg/ml BSA, under mineral oil at 39°C, 5% CO2 in humidified air during 24 h. IVF in semen samples was evaluated by cleavage rates.

Statistical analysis

Data were expressed as mean ± SEM. Values were compared using Analysis of Variances (ANOVA). A p-value < 0.05 was considered significant.

Results

As regards routine tests, no significant differences were observed in progressive motility and vigor between samples of animals with high and low fertility in vivo, but differences were detected in vitality and acrosomal integrity (p<0.05) (Figure 1). Within functional tests, no differences were detected in thermoresistance (data not shown) and capacitation induction, but HOS test, acrosomal reaction induction and IVF showed significant differences (p<0.05) (Figure 2).

Citation: Morado S, Pereyra V, Breininger E, Sara R and Cetica P. Study of Sperm Evaluation Parameters to Estimate Cryopreserved Bovine Semen Fertility. Austin J Vet Sci & Anim Husb. 2015;2(1): 1005. ISSN:2472-3371