Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

doi:10.1016/j.theriogenology.2011.05.012

Theriogenology

Volume 76, Issue 6, 1 October 2011, Pages 1070-1075

https://doi.org/10.1016/j.theriogenology.2011.05.012 Get rights and content

Abstract

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. The aim of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro. In vitro matured bovine oocytes were inseminated with fresh semen stored in a caprogen-based diluent, with or without prior Percoll separation. Irrespective of sire, storage of fresh semen at ambient temperature for up to 7 d post collection had no effect on cleavage rate or blastocyst development after IVF. In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Higher numbers of fresh sperm migrated through artificial mucus on Day 0 (day of semen collection) compared with frozen-thawed sperm. On Day 1 and 2 postcollection there was no difference in the number of sperm migrating through the mucus, but storage of sperm at ambient temperature for longer than 2 d resulted in a significant decline in their ability to penetrate mucus compared with frozen sperm from the same ejaculate. In conclusion, bovine sperm retain the ability to fertilize oocytes in vitro for up to 7 d following storage at ambient temperature. However, the ability of sperm to migrate through artificial mucus in vitro is severely depressed after 2 d storage which may have significant implications for the ability of these sperm to reach the site of fertilization in vivo after AI.

Introduction

Artificial insemination is the method of choice for dissemination of genetic improvement. The use of fresh semen for AI enables a 10-fold reduction of the insemination dose compared with frozen-thawed semen without a reduction in pregnancy rates [1], [2] thus allowing the more widespread use of high genetic merit sires. Recent world statistics for AI in cattle stand at 232 million doses of frozen and 11.6 million of fresh semen, the latter restricted primarily to New Zealand with smaller amounts used elsewhere [3]. The process of freezing and thawing irreversibly affects a significant proportion of sperm with typical estimates of 50% survival postthawing.

The potential genetic contribution of a sire is determined by the number of progeny he leaves and his genetic superiority. In cattle, the number of progeny is determined by (1) the total sperm output of the bull, (2) the number of sperm used per insemination, and (3) the percentage of cows calving to a single insemination. The average sperm production of a bull ranges from approximately 4 to 12 × 10⁹ sperm per ejaculate. Typically, when using liquid semen, a minimum number of 2.5 × 10⁶ total sperm per dose is required. Consequently, each ejaculate could provide up to 4800 doses. However, when using frozen semen, approximately 20 million total sperm per dose are required, amounting to a maximum of about 600 doses of frozen semen per ejaculate. On average, the number of semen doses possible for a single sire in a calendar year ranges between 100 000 and 150 000 if used exclusively as frozen semen. However, the seasonal mating patterns in some countries such as New Zealand, Australia, and Ireland, allows distribution of semen in liquid form. The advantages of using fresh semen include the ability to use low sperm numbers per insemination, high sire utilization, inexpensive storage, and ease of use in the field. However, this form of storage lacks the flexibility of frozen semen due to its finite shelf life [2], [4].

Storage of bovine semen in the fresh and frozen state has been comprehensively reviewed [2]. Bovine spermatozoa stored at ambient temperature in Caprogen diluent show a slow decrease in motility over an extended period about 3 to 4 wk, but the associated decrease in fertility is significantly higher [1]. Fertility, as measured by nonreturn rate, is maintained for the first 3 to 5 d after dilution, followed by a rapid decrease in non return rate until Day 10 of storage [1]. The physiological reasons for this rapid decline in fertility of spermatozoa stored at ambient temperature is presumed to be due to extracellular oxidative stress, effects of seminal plasma, and endogenous free radical production [1].

Although the use of fresh semen in the Irish dairy AI industry only accounts for 5% of total AI usage, this may peak to over 25% during the spring breeding season due to the increased demand for Irish proven sires of high genetic merit. With such sires, this can result in insufficient semen being available during periods of highest demand. Currently, in Ireland, fresh semen is only used for 2 d postcollection due to the perceived drop in pregnancy rates subsequently. The ability to use fresh semen at low dose rates over a period of 3 to 4 d would significantly improve the number of inseminations per ejaculate per bull.

The objective of this study was to examine the effect of storage of fresh semen for up to 7 d at ambient temperature on fertilization and embryo development in vitro, and on the ability of sperm to penetrate artificial mucus in vitro.

Section snippets

Semen collection and processing

Semen from Holstein Friesian bulls standing at an AI center was used throughout (National Cattle Breeding Centre, Enfield, Ireland). Semen was collected by artificial vagina and split for conventional freezing or fresh processing. Fresh semen was collected and diluted to 20 × 10⁶ sperm/mL in Caprogen diluent, stored at room temperature and used to fertilize in vitro matured (IVM) oocytes at 5 to 8 h post semen collection (the time taken to collect, process and transport the semen to the IVF

Experiment 1: effect of duration of storage of fresh bovine semen on embryo development in vitro following IVF

There was no bull effect (P > 0.05) or duration of storage effect (P > 0.05) on oocyte cleavage rate or blastocyst development after IVF. Irrespective of sire, storage of fresh semen at ambient temperature for up to 4 d postcollection had no effect on cleavage rate or blastocyst development after IVF with Percoll-separated sperm (Fig. 1). In addition, blastocyst quality, as assessed by the proportion of blastocysts hatching from the zona, was not affected by semen storage. Similarly, when IVF

Discussion

The main findings of this study are (1) fresh bovine sperm stored at room temperature are capable of fertilizing oocytes in vitro for at least 7 d without any reduction in development, and (2) storage of fresh sperm at room temperature for longer than 2 d results in a significant reduction in their ability to penetrate artificial mucus.

In contrast to natural mating in cattle, during which approximately 10 × 10⁹ sperm are deposited in the anterior vagina, AI bypasses the cervical barrier,

Acknowledgments

The authors thank the staff of the National Cattle Breeding Centre, Enfield, County Meath, Ireland, for provision of semen and to the staff of Kildare Chilling, County Kildare, Ireland, and Kepak, Clonee, County Meath, Ireland, for allowing access to bovine ovaries. J.I. Marti was supported by a grant from Programa Europa CAI-CONSI+D. A. Al Naib was funded by the Syrian University of Aleppo.

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Cited by (12)

Comparison of pregnancy outcomes using either an Ovsynch or a Cosynch protocol for the first timed AI with liquid or frozen semen in lactating dairy cows
2018, Theriogenology
Citation Excerpt :
This issue might be resolved by using liquid semen for AI. Liquid semen has compelling advantages such as reduced insemination doses [5,6], less damage to sperm [7], more widespread use of genetically superior sires, limiting the negative effect of sexing technology [6], lower cost of storage, and simple handling [8]. In contrast, the major disadvantage for liquid semen is its limited fertile life span of approximately 3 days [8].
The objective of this study was to evaluate fertility to the first timed AI (TAI) using either liquid semen or frozen semen after an Ovsynch or a Cosynch protocol in lactating dairy cows. The hypothesis was that there is an increase in fertility to the first TAI when cows are inseminated with liquid semen compared to that when frozen semen is used in a Cosynch protocol. Lactating dairy cows (n = 1724; 540 primiparous, 1184 multiparous) from 9 commercial dairy farms were enrolled on a weekly basis to facilitate first timed AI. Two experiments were conducted. In experiment 1, all cows received GnRH, 7 d later PGF_2α, and then received one of the following treatments: 1) GnRH + TAI with liquid semen 56 h after PGF_2α; 2) GnRH + TAI with frozen semen 56 h after PGF_2α; 3) GnRH 56 h after PGF_2α + TAI with liquid semen 12–16 h after the second GnRH; 4) GnRH 56 h after PGF_2α + TAI with frozen semen 12–16 h after the second GnRH. In experiment 2, all cows received GnRH, 7 d later PGF_2α, and then received treatments 3 or 4 as described for experiment 1. Number of sperm per straw was 20 × 10⁶ sperm/straw and 10 × 10⁶ sperm/straw for frozen and liquid semen, respectively. Pregnancy diagnosis was performed by ultrasound scanning at 39 d after TAI. In experiment 1 (n = 1263), there was an interaction of semen preservation method by TAI protocol. Cows inseminated with liquid semen concurrently with the second GnRH (Cosynch-56) achieved greater pregnancy per AI (P/AI) than cows inseminated with frozen semen using the same synchronization protocol (20.0% vs. 27.5%; P = 0.032). There was no effect of semen preservation method (liquid semen 32.3% vs. frozen semen 28.6%; P = 0.330) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (34.8% vs. 20.2%; P = 0.001). In experiment 2 (n = 377), there was no effect of semen preservation method (liquid semen 26.5% vs. frozen semen 25.5%; P = 0.846) when cows were inseminated approximately 16 h after the second GnRH injection (Ovsynch-56). Parity affected P/AI with primiparous having a greater P/AI than multiparous cows (37.0% vs. 17.3%; P = 0.001). The results of this study provide evidence that liquid semen achieved greater P/AI in a TAI protocol with a long time interval between insemination and ovulation (Cosynch-56) compared with frozen semen indicating that liquid semen might have a longer viability in the reproductive tract.
Relationship between in vitro sperm functional assessments, seminal plasma composition, and field fertility after AI with either non-sorted or sex-sorted bull semen
2017, Theriogenology
Citation Excerpt :
Bulls were selected on the basis of the greatest difference between pregnancy rates from a cohort of Holstein Friesian bulls who were stationed in Ireland, had greater than 200 inseminations, and were still alive. Pregnancy rate was defined as pregnancy to a given service identified retrospectively either from a calving event and/or where a repeat service (or a pregnancy scan) deemed the animal not to be pregnant to the said service [37,38]. Cows/heifers which were culled or died on farm were omitted.
The hypothesis of this study was that different in vitro parameters are required to predict the in vivo fertility of non-sorted (NS) and sex-sorted (SS) semen. Thus, the aim was to correlate in vitro bull sperm functional parameters (experiment 1) and seminal plasma composition (experiment 2) with pregnancy rates using 2 cohorts of bulls (NS and SS). Experiment 1: ejaculates from each bull (n = 3 ejaculates per bull; n = 6 bulls for both NS and SS) were assessed for motility, thermal stress tolerance and morphology using microscopy, and viability, osmotic resistance, mitochondrial membrane potential, and acrosome integrity using flow cytometry. Fertilizing ability was assessed using IVF. Experiment 2: ejaculates (n = 3 per bull; n = 8 and 6 bulls for NS and SS, respectively) were collected, seminal plasma harvested and frozen and later analyzed for amino acid and fatty acid composition using gas chromatography mass spectrometry. In the NS cohort of bulls, there was no correlation between pregnancy rate and any of the sperm functional parameters assessed. However, within the SS cohort, motility and viability were correlated with pregnancy rate (r = 0.84 and 0.80, respectively; P < 0.05). There was no correlation between IVF outcome and pregnancy rate in either the SS or NS cohort of bulls. In the NS cohort of bulls, concentrations of the amino acid isoleucine and the fatty acid tricosylic acid (C23:0) were correlated with pregnancy rate (r = 0.80 and 0.74, respectively; P < 0.05). Within the SS cohort of bulls, the amino acid glutamic acid and the fatty acid arachidic acid (C20:0) were correlated with pregnancy rate (r = 0.84 and 0.82, respectively; P < 0.05). In conclusion, this study suggests that different in vitro markers of fertility are required to predict the fertility of NS and SS sperm.
Property profiling of biosimilar mucus in a novel mucus-containing in vitro model for assessment of intestinal drug absorption
2014, European Journal of Pharmaceutics and Biopharmaceutics
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Mucus mixtures have been used in several other studies with varying composition. Often, simple single component solutions of mucin or hyaluronic acid [26] are employed as models of mucus, whereas the use of more complex mixtures as for example mucin mixed with diethylene triamine pentaacetic acid, egg yolk and DNA is also reported [27]. Recently, Teubl et al. used a disk of mucin and glycerol to introduce mucus to the surface of the buccal TR146 cell culture model [28].
Oral delivery of drugs, including peptide and protein therapeutics, can be impeded by the presence of the mucus surface-lining the intestinal epithelium. The aim of the present project was to design and characterize biosimilar mucus compatible with Caco-2 cell monolayers cultured in vitro to establish a more representative in vitro model for the intestinal mucosa. The rheological profile of a biosimilar mucus mixture composed of purified gastric mucin, lipids and protein in buffer was optimized by supplementing with an anionic polymer to display viscoelastic properties and a microstructure comparable to freshly isolated porcine intestinal mucus (PIM). Further, this multicomponent biosimilar mucus mixture was optimized with regard to the lipid content in order to obtain cellular compatibility with well-differentiated Caco-2 cell monolayers. In contrast, PIM was found to severely disrupt the Caco-2 cell monolayer. When combined with the Caco-2 cell monolayers, the final biosimilar mucus was found to significantly affect the permeability profiles for hydrophobic and hydrophilic small and large model drug compounds in different ways. In conclusion, the present study describes an improvement of the biorelevance of the Caco-2 cell culture model by application of mucus, resulting in an in vitro model of oral mucosa suitable for future assessment of innovative drug delivery approaches.
Improving bovine semen diluents: Insights from the male and female reproductive tracts, and the potential relevance of cervical mucins
2014, Animal
The commercial applicability of bovine artificial insemination (AI) depends on the effectiveness of diluents for maintaining sperm fertility. Challenges faced by the AI industry due to recent advances in assisted reproduction, and the limitations inherent in using fresh and frozen-thawed sperm for AI, could be overcome with the development of better semen diluents. Research into the different microenvironments of bovine sperm as they progress towards maturity, capacitation and fertilisation is revealing various mechanisms that could be exploited to improve the formulation of semen diluents. These are reviewed here. A rationale for a more detailed investigation of bovine cervical mucus for factors that may allow further progress towards this goal are also discussed.
Reducing sperm concentration is critical to limiting the oxidative stress challenge in liquid bull semen
2013, Journal of Dairy Science
Citation Excerpt :
In contrast, other studies have reported that human liquid semen samples with higher levels of ROS had impaired sperm motility (Iwasaki and Gagnon, 1992; Armstrong et al., 1999; El-Taieb et al., 2009). In addition, it was recently reported that sperm stored for greater than 3 d have a reduced ability to penetrate artificial mucus (Al Naib et al., 2011b); however, the assessment of mitochondrial activity in the current study showed that the number of live sperm with active mitochondria remained stable and high throughout the study (ranging from 94.8 ± 0.53% to 91.9 ± 0.63%). This indicates that the inability of stored sperm to penetrate artificial mucus is not attributable to sperm mitochondrial activity.
Because of the short breeding season, the use of liquid bull semen is a viable option in seasonal grass-based dairy systems such as Ireland. Currently in Ireland, liquid bull semen contains approximately 5 million sperm per insemination dose and is used within 2.5 d of collection. The hypothesis of this study was that reducing the sperm number per insemination dose would enable bull sperm to be stored for longer. Semen was collected at a commercial AI center and diluted to 1 (T1), 2 (T2), 3 (T3), 4 (T4), and 5 (T5) million sperm per 0.25-mL dose in caprogen diluent. On d 0.25 (6 h postcollection), 1, 2, 3, 4, and 5 postcollection, viability, oxidative stress, and mitochondrial activity were assessed using flow cytometry and the fluorescent probes propidium iodide, CM-H₂DCFDA, and rhodamine 123, respectively. On the same days, glucose consumption, total antioxidant capacity, and progressive linear motility were assessed. We observed an effect of day and treatment on sperm cell viability, with the highest percentage live found in T 0005 and the lowest in T 0025 on all days. Oxidative stress in live sperm increased with duration of storage and was affected by treatment, being highest in T 0025 and lowest in T 0005 on all days (d 5: 56.4 ± 2.76% and 28.8 ± 1.22%, respectively; mean ± SEM). Both the total antioxidant capacity and percentage of live sperm positive for rhodamine 123 were unaffected by treatment. The concentration of glucose in caprogen declined with time and was lowest in T 0025 and highest in T 0005 on d 5. In conclusion, higher concentrations of sperm have detrimental effects on sperm cell viability and increase oxidative stress but have no effect on the mitochondrial activity of sperm.
Effect of Adding α-tocopherol on Fertility Parameters in Bovine Semen Cryopreservation
2023, Agricultural Science Digest

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Research articleEffect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm

Abstract

Introduction

Section snippets

Semen collection and processing

Experiment 1: effect of duration of storage of fresh bovine semen on embryo development in vitro following IVF

Discussion

Acknowledgments

Anim Reprod Sci

Theriogenology

Anim Reprod Sci

Theriogenology

Anim Reprod Sci

Theriogenology

Theriogenology

Theriogenology

Theriogenology

Research article
Effect of duration of storage at ambient temperature on fertilizing ability and mucus penetration ability of fresh bovine sperm