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Contents

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

With the advancement of assisted reproductive biotechnologies, preselecting the sex of offspring has become an important goal for cattle and other livestock breeding as well as for research. The aim of this study was to investigate the feasibility of producing enriched pools of X- or Y-chromosome-bearing sperm by vertical swim-up through a long, narrow column. Sperm recovered from the top portion of the column was predominantly Y-bearing (60%, p < 0.05), which were capable of fertilizing matured oocytes and produce significantly more male embryos compared with standard swim-up protocol.


Introduction

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Difference in the sex-chromosome constitution is the base of sex determination in mammals, with females having two X-chromosomes and males having a X- and a Y-chromosome. Therefore, the male gamete determines the genetic sex of an offspring at fertilization because spermatogenesis results in two genetically distinct populations of spermatozoa, one bearing the X-chromosome and the other the Y-chromosome. Due to evolutionary specialization of the Y-chromosome, it has become smaller than the X, in most species, and this difference can be precisely measured in the DNA content by flow cytometry. This technique has provided the basis for the patented sperm sorting technology (FACS), capable of producing offspring of the desired sex (Johnson and Welch 1999). Many other differentiating properties of the two sperm populations, such as size, motility, longevity, surface charge and antigen profile, have been also explored in attempts to separate X- and Y-chromosome-bearing spermatozoa for the advancement of animal breeding, but results have been controversial (Hamano 2007). It should be noted that unknown physiological conditions could also affect the natural ratio of X- and Y-bearing spermatozoa in semen, as Chandler et al. (1998) have found considerable variation in the sex ratio of spermatozoa and of calves produced from different ejaculates of AI bulls.

Sperm swim-up is a technique routinely used to create pools of sperm enriched with highly motile, viable sperm for in vitro fertilization. In this protocol, semen is layered under 1–2 ml of sperm-TALP (Tyrode's albumin lactate pyruvate) for variable duration (30–60 min) of incubation after which the top most fraction is aspirated and used for in vitro fertilization (Holt et al. 2010); however, the procedure does not alter the ratio of X- and Y-chromosome-bearing sperm (Yan et al. 2006; Orsztynowicz et al. 2013).

Here, we provide information on a modified vertical swim-up method carried out in a long narrow tube that challenges the swimming ability of bull spermatozoa. This technique resulted in a motile sperm population enriched in Y-chromosome-bearing cells in the upper portion of the swim-up tube. The spermatozoa obtaining by this method were capable of fertilizing oocytes in vitro and preferentially producing male embryos.

Materials and Methods

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

Sperm preparation

Frozen semen from three bulls (bull A, B, C) of known good reproductive health and field fertility was used for this study. The standard swim-up procedure, the control, was carried out by depositing 0.5 ml semen (15 × 106 sperm) under 1.5 ml of Sperm-TALP medium in 4-ml tubes incubated in vertical position at 37°C for 45 min. Pipette-swim-up (test group) was carried out in 5-ml (30 cm long) plastic serological pipettes filled with equilibrated Sperm-TALP and placed in a rubber stopper (Fig. 1). Subsequently, 0.25 ml semen (7.5 × 106 cells) was carefully injected through the rubber into the bottom of the pipette using a syringe. In a preliminary experiment, sperm from bull A was incubated in a pipette for 10, 20, 30, 40 and 45 min. The bottom portion of the pipette was discarded, and the remaining eight 1 ml zones (3.5 cm height/zone) were individually emptied from the bottom of the pipette into tubes. Sperm concentration was determined using a hemocytometer. Later spermatozoa from all three bulls were subjected to three independent experiments of standard and pipette-swim-up for 45 min, and the 7th and 8th (top) fractions were collected.

image

Figure 1. The pipette-swim-up column was created from a regular 5-ml serological pipette and a rubber stopper. Arrows mark the fractions collected

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Sexing of spermatozoa by FISH

Smears were made from untreated, control and zones 7 and 8 test groups of sperm and were decondensed by treating the slides in 0.2 m Tris (pH 8.6, Sigma, St. Louis, MO, USA) solution containing 1.25% papain (Sigma) and 0.16% DTT (Sigma) for 2 min. The steps of fluorescent in situ hybridization using X- and Y-chromosome-specific painting probes (Fig. 2, Cambio, Cambridge, UK) were previously described in detail (Revay et al. 2002). All types of samples (untreated, control swim-up and pipette-swim-up) were made from the mixture of two straws, and the swim-up experiments were repeated three times. The chi-squared test was used for statistical analysis.

image

Figure 2. Representative image of the X–Y-sperm-FISH in Bull A. The X-chromosome is labelled green and the Y-chromosome is red

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In vitro embryo production and embryo sexing

In vitro oocyte maturation (IVM) and in vitro fertilization (IVF) were carried out according to Ashkar et al. (2010). Briefly, cumulus–oocyte complexes were aspirated from ovarian follicles, matured in 80 μl drops of TCM199 medium (2% steer serum, 1 μg/ml estradiol, 0.5 μg/ml bFSH, 1 μg/ml bLH) in 5% CO2 at 38.5°C for 20–22 h. Either 1 × 106/ml standard swim-up sperm or 0.1 × 106/ml pipette-swim-up sperm from zone eight from only Bull A was used for IVF in three independent trials. Presumptive zygotes were stripped of their cumulus cells by vortex after 18–20 h post-insemination and then cultured in groups of 30 in 30 μl drops of synthetic oviduct fluid medium for 48 h. The zona pellucida was removed using 0.2% pronase from the collected 2- to 8-cell stage embryos (n = 100), then individually lysed and sexed by PCR amplifying the Y-chromosome-specific TSPY and autosomal control GAPDH genes according to Hamilton et al. (2012).

Results

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

We observed spermatozoa in zones 1–4, 5, 6, 7, 7–8 of the pipette after 10, 20, 30, 40 and 45 min of incubation, respectively. The calculated average sperm swim-up speed (distance/time) was 93 μm/s. The sperm concentration progressively decreased towards higher zones with an average concentration falling from 5.8 × 106 sperm/ml in zone 1 to 0.1 × 106 sperm/ml at zone 8 (Table 1).

Table 1. Bull A sperm concentration collected from different pipette-swim-up zones
Swim-up zoneConcentration (×106 sperm/ml)Average concentration (×106 sperm/ml)Std Dev
16.35.45.75.80.46
21.71.61.01.40.38
30.80.80.90.80.06
40.30.90.10.40.42
50.50.30.10.30.20
60.40.30.30.30.05
70.20.20.30.20.06
80.080.20.10.10.06

FISH sexing of untreated and control swim-up spermatozoa from three bulls revealed no difference from the expected 50 : 50 ratio; however, the examination of more than 20 300 sperm from zones 7 and 8 showed enrichment of Y-bearing sperm (60 : 40, p < 0.05, Table 2).

Table 2. Results of FISH sexing in 3 bulls using spermatozoa from zone 7 and 8
BullX-Y FISHControl (%)Zone 7 (%)Zone 8 (%)
AY481 (47.2)2515 (60.9)2256 (59.4)
X538 (52.8)1614 (39.1)1542 (40.6)
BY648 (51.5)986 (57.2)3723 (59.2)
X610 (48.5)737 (42.8)2566 (40.8)
CY329 (53.1)1306 (61)1331 (58.6)
X291 (46.9)835 (39)940 (41.1)

The cleavage rate (assumed to correlate with fertilization rate) in the oocyte groups fertilized with sperm from Bull A after pipette-swim-up was lower than with sperm after control swim-up (49% vs 76%, p < 0.05); however, the sex ratio in the group fertilized by pipette-swim-up sperm was significantly skewed towards males (62/38% vs 47.5/52.5%, p < 0.05, Table 3).

Table 3. Cleavage rate and sex ratio of in vitro fertilization (IVF) embryos produced from pipette-swim-up separated spermatozoa from Bull A
MethodNumber of oocytesNumber of oocytes cleaved (%)Sex ratio (male : female%)
  1. a

    X2, p = 0.046.

Pipette-swim-up20299 (49%)62 : 38a
Control141107 (76%)47 : 53

Discussion

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References

In this study, we demonstrated the ability of bull sperm to swim-up along vertical Sperm-TALP column up to 30 cm in 45 min (Table 1), and those reaching the top portion were significantly enriched in Y-chromosome-bearing cells. This is demonstrated in the sperm population by FISH (~60% Y-bearing) and also by the similar skewing of the sex ratio towards male embryos (62%) following IVF (Tables 2 and 3). There was a noticeable lower cleavage rates in the embryos fertilized by pipette-swim-up sperm, compared with the control (49% vs 76%). It is known that the fertilization rate in vitro is influenced by the concentration of sperm (Holt 2000), which in the present study was only approximately 10% of control. However, it is not known whether the cleavage rate decrease noted here was solely due to the lower sperm concentration or due to other confounding factors associated with the pipette-swim-up. Nonetheless, fertilization, albeit at a lower rate, was possible with low sperm concentration subsequent to pipette-swim-up.

As the fastest moving sperm (top fraction) was predominantly Y-chromosome bearing, we speculate that under the given in vitro conditions, the Y-chromosome-bearing spermatozoa swim faster than X-bearing ones. This otherwise frequently held view was not supported by Penfold et al. (1998), who compared the motility of sorted X- or Y-bearing sperm samples in salt solution and found no differences in velocity, although linearity and straightness were favoured in X-sperm. Measurement of these parameters was not our aim, instead the observation of the composition of a native (non-altered) sperm population under challenging swimming condition and to study its effect on IVF embryos. Previous studies also support our observations of embryo sex ratio by showing that reduction of the length of IVF from 18 h to 6 h resulted a significant decrease in the fertilization rate but an increase in the percentage of male embryos (Kochhar et al. 2003), suggesting the Y-bearing sperm can fertilize the oocytes faster in vitro. In contrast, pre-incubation of sperm for 24 h before co-incubation with in vitro-matured oocytes resulted in a significant decrease in the percentage of males (Lechniak et al. 2003). Taken together, these observations suggest that X-and Y-bearing bull spermatozoa swim at different speeds and have different windows of opportunity for fertilization.

In summary, this study showed that swim-up through a narrow vertical column generated Y-chromosome enriched bull sperm that was capable of fertilizing oocytes and producing IVF embryos with higher proportion of males.

Acknowledgement

This research was funded by the Canada Research Chairs program, the Department of Biomedical Sciences and the Ontario Veterinary College, University of Guelph.

Author contributions

A. Azizeddin carried out the swim-up and IVF procedures and took part in the analysis of results. F. Ashkar contributed to the study design and helped with the in vitro embryo production. WA King contributed to the conception and design of the study, drafting and critical revision of the manuscript. T. Revay carried out the sperm-FISH experiments, contributed to the study design, data analysis and drafting the manuscript.

Conflict of interest

The authors have no conflict of interest to disclose.

References

  1. Top of page
  2. Contents
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. References