Research Article: Impact of proximal cytoplasmic droplets on quality traits and in-vitro embryo production efficiency of cryopreserved bull spermatozoa

Date Published: January 12, 2012

Publisher: BioMed Central

Author(s): Janaina T Carreira, Gisele Z Mingoti, Lucia H Rodrigues, Carlos Silva, Silvia HV Perri, Marion B Koivisto.


Proximal cytoplasmic droplets (PCDs), a remnant of germ cell cytoplasm, are common non-specific morphological defects in bovine semen. This study evaluated the effect of higher percentages of PCDs on the quality of frozen-thawed bovine semen, embryo production and early embryo development.

Three ejaculates from each of five (group 1: PCD ≤ 1%, control) and eight adult Bos indicus bulls (group 2: PCD ≥ 24%) were analysed. Semen samples were examined for: post-thaw motility, vigour of movement, concentration, sperm morphology, slow thermoresistance test (STT), membrane integrity, acrosome status, mitochondrial function using fluorescent probes association (FITC-PSA, PI and JC-1) and sperm chromatin integrity using acridine orange assay. Two bulls from group 2, with 28.5% and 48.5% PCD, respectively, and three bulls from the control group, each with 0% PCD, were selected for IVF (in vitro fertilisation).

Semen analyses revealed a significant correlation (P < 0.01) between increased rates of PCD and sperm quality traits. Nevertheless, no differences were observed in sperm motility and vigour either before or after the STT or in the percentage of intact acrosomes (analysed by differential interference contrast microscopy (DIC) after STT), but membrane integrity, acrosome status (evaluated with FITC-PSA staining method after thawing) and mitochondrial function were reduced, when compared with group 1 (P < 0.05). The higher incidence of PCD was positively correlated to chromatin damage, especially after three hours of incubation at 37°C. IVF showed similar results for bull C2 (group 1, control) and bull P2 (group 2, group with higher PCDs). Higher PCD levels influenced spermatozoa quality traits. IVF and embryo development data showed that cleavage, blastocyst formation and blastocyst hatching may have been influenced by the interaction of morphology traits and individual bull effects.

Partial Text

In the last decade, major advances have been made in the understanding of the biochemical and molecular mechanisms that determine the production of functionally competent spermatozoa [1]. Disturbed sperm-oocyte interaction is the principal cause of low IVF rates in humans and seems to be more associated with sperm defects than with oocyte defects because sperm can penetrate the zona pellucida of oocytes at any stage of maturity and quality. Thus, the proportion of zona pellucida-bound spermatozoa with normal or abnormal morphology is strongly related to IVF success rates [2].

The null hypothesis (H0) was that there were no differences between bulls with high and low levels of PCD regarding sperm quality traits and embryo production efficiency. Statistical analyses were performed using the Statistical Analysis System software (release 9.2. SAS Institute Inc., Cary, NC, USA, 2008). Data (except vigour) were transformed in arcsin% to obtain a normal distribution. Concentration, PIA, membrane integrity, acrosome integrity, mitochondrial potential and IVF results were examined using two-way ANOVA (bull × group); for the STT, results (motility, vigour and DNA integrity) were tested by MANOVA (bull × group × incubation time). The means were compared using least square methods. The correlation coefficient (r) and determination factor (r2) were calculated for motility, vigour, membrane and acrosome integrity, mitochondrial potential and DNA integrity. The data were presented as means ± SDs and were considered statistically significant when P < 0.05 [24]. The post-thaw semen quality of the five bulls from group 1 and eight bulls of group 2 are summarised in Tables 1 and 2. No statistical differences were seen between the groups for sperm concentration and the percentage of minor morphological defects; nevertheless, as expected, total major defects and the percentage of PCDs were higher for group 2 - PCD (P < 0.05) (Table 1). The total morphological defects percentages can be mainly attributed to the higher PCD levels as seen by the determination factor (0.93) and the regression equation (total morphological defects = 11.69 +1.41PCD). Previous studies have related spermatozoa with increased percentages of PCDs to lower motility in humans [25]. A similar connection was found in the present study, as seen by the significant (P < 0.001) negative correlation between motility and PCDs. In young beef bulls, zero to six weeks after puberty, the percentage of progressively motile spermatozoa increased rapidly, while the percentage of spermatozoa with proximal cytoplasmic droplets was correspondingly dramatically reduced [26]. High percentages of ejaculated spermatozoa with retained cytoplasmic droplets are associated with infertility in adult bulls [3,8,9]. A recent review indicated that elimination of the droplets during ejaculation may be prognostic for fertility, while their retention may indicate sub- or infertility [27]. This study indicated that higher levels of PCDs could influence the sperm quality traits; nevertheless, this effect was not sufficient to impair motility and vigour, but it did affect the integrity of the acrosomal membrane and plasma membrane and resulted in low mitochondrial potential after thawing. Chromatin was not affected overall by higher levels of PCDs, but it showed a greater correlation with damage, especially after 3 h of incubation at 37°C. As previously stated, IVF and embryo production results may have been affected by the interaction of morphology traits and individual bull effects. The authors declare that they have no competing interests. JTC carried out the semen evaluation and IVF procedures and wrote the manuscript; GZM coordinated the IVF procedure; LHR and CR participated in semen collection, freezing and evaluation; SHVP performed the statistical analyses; and MBK was the organiser and the principal investigator. All the authors have read and approved the final manuscript.   Source: