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Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows Michiko Noguchi a,1 , Koji Yoshioka a,∗ , Hirokazu Hikono a , Chie Suzuki a , Kazuhiro Kikuchi b a b
National Institute of Animal Health, Tsukuba, Ibaraki, Japan National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
a r t i c l e
i n f o
Article history: Received 13 June 2015 Received in revised form 29 August 2015 Accepted 2 November 2015 Available online xxx Keywords: Adenosine Deep intrauterine insemination Frozen-thawed semen Pig Theophylline
a b s t r a c t We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6 h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6 h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5 × 108 frozen-thawed spermatozoa by DIUI at 34 h after the hCG injection. At 4 h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10 × 108 frozen-thawed spermatozoa at 36 h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Artificial insemination (AI) techniques for pig breeding have been focused on reducing the number of
∗ Corresponding author at: National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan. E-mail address: [email protected] (K. Yoshioka). 1 Present address: Laboratory of Theriogenology, Department of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
spermatozoa per dose and/or determining optimal AI timing during estrus while maintaining good reproductive performance (reviewed by Roca et al., 2006b). Successful fertilization by AI in pigs depends on the number of spermatozoa in the uterotubal junction (UTJ) around the time of ovulation (Krueger et al., 1999). Approximately 20–45% of the spermatozoa inseminated into the cervix are lost as they flow back out of the reproductive tract (Steverink et al., 1998; Matthijs et al., 2003). The technique of deep intrauterine insemination (DIUI) can diminish the backflow of semen from the genital tract (Martinez et al.,
http://dx.doi.org/10.1016/j.anireprosci.2015.11.006 0378-4320/© 2015 Elsevier B.V. All rights reserved.
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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2001, 2002). Furthermore, farrowing rates and litter sizes in sows after DIUI with 1000 × 106 frozen-thawed spermatozoa are equivalent to those of sows inseminated with 6000 × 106 frozen-thawed spermatozoa into the cervix (Roca et al., 2003). Therefore, despite the poor condition of frozen-thawed spermatozoa compared with those of fresh semen (Penã et al., 2003; Roca et al., 2006a), they have been used in DIUI as an efficient tool for piglet production (Roca et al., 2003; Wongtawan et al., 2006). When sows are inseminated into the cervix semen backflow and sperm cell phagocytosis by polymorphonuclear leukocytes (PMNs) leads to loss of spermatozoa in the uterus. It has been reported that migration of PMNs into the uterus and subsequent sperm phagocytosis depends on the composition of the semen extender used in pigs (Matthijs et al., 2003; Yamaguchi et al., 2009). Methylxanthines, especially caffeine, stimulate both capacitation and spontaneous acrosome reaction of boar spermatozoa in vitro (Funahashi et al., 2000a; Funahashi and Nagai, 2001). In vivo experiments showed that caffeine could suppress the intrauterine migration of PMNs when pigs were artificially inseminated with fresh or frozen-thawed semen diluted with Beltsville thawing solution (BTS) supplemented with caffeine and calcium chloride (CaCl2 ) (BCC) (Matthijs et al., 2003; Yamaguchi et al., 2009). Because of this inhibition of PMN migration with caffeine, the numbers of spermatozoa in the oviducts, and the pregnancy and farrowing rates produced by frozen-thawed semen diluted with BCC were higher than those of semen diluted with Modena solution, which does not contain caffeine (Yamaguchi et al., 2009). Moreover, the addition of caffeine to the modified Modena solution for thawing enhanced the motion quality parameters of frozen-thawed boar sperm with no influence on the integrity of plasma or acrosomal membranes (Yamaguchi et al., 2013). These results indicate that caffeine supplementation of semen extenders can enhance sperm transportation into the UTJ and improve reproductive performance in vivo. Adenosine, which is present in the female reproductive tract, stimulates capacitation and inhibits spontaneous acrosome reactions in boar spermatozoa (Funahashi et al., 2000a,b; Funahashi and Nagai, 2001). Adenosine supplementation of in vitro fertilization (IVF) medium can produce better sperm penetration rates than methylxanthine supplementation (Funahashi et al., 2000b; Funahashi and Nagai, 2001; Yoshioka et al., 2003). These reports suggest that supplementation with adenosine in semen extenders might enhance reproductive performance after DIUI with spermatozoa with low quality and/or in a small volume. The objectives of this study were to determine the effects of IVF medium supplemented with methylxanthine and adenosine as a semen extender on (1) sperm characteristics, (2) distribution of spermatozoa in the female genital tract and (3) reproductive performance in sows after DIUI. 2. Materials and methods 2.1. Animals All animal-related procedures followed in this study were approved by the Institutional Care and Use
Table 1 Components of the semen extenders used. Component
mMS
PFM
NaCl (mM) KCl (mM) KH2 PO4 (mM) MgSO4 ·7H2 O (mM) NaHCO3 (mM) Glucose (mM) Na-pyruvate (mM) Ca-(lactate)2 ·5H2 O (mM) Theophylline (mM) Adenosine (M) l-cysteine (M) Gentamicin (g/mL) PVA (mg/mL) Na-citrate·2H2 O (mM) Citric acid (mM) EDTA·2Na (mM) Tris (mM)
– – – – 11.90 152.61 – – – – – 10 – 23.46 15.10 6.99 46.66
108.0 10.0 0.35 0.40 25.07 1.00 0.2 4.0 2.5 1.0 0.25 10 3.0 – – – –
mMS, modified Modena solution; PFM, porcine fertilization medium.
Committee for Laboratory Animals of the National Institute of Animal Health in Japan (Protocol No. 11-003). Animals were kept in individual crates with water available ad libitum and received 2.2 kg of a commercial diet once daily. Estrus detection in sows was performed twice daily using a mature boar. Estrus was defined as the period during which sows exhibited a standing response for the boar. 2.2. Preparation of frozen-thawed boar spermatozoa and semen extenders The method of semen cryopreservation was essentially the same as that described by Yoshioka et al. (2003) with minor modifications. Briefly, the collected semen was diluted 1:2 with Modena solution supplemented with 10g/mL gentamycin (mMS; Table 1) and kept overnight at 15 ◦ C. After centrifugation at 800 × g for 10 min, the precipitated spermatozoa were resuspended at a density of 2 × 109 cells/mL in the first diluent (0.3 M lactose solution containing 20% egg yolk, 75 mg/mL amikacin, 25 mg/mL dibekacin and 2,500,000 IU/mL potassium penicillin G) and then cooled to 5 ◦ C for 2–3 h. The samples were then further diluted 1:1 with the second diluent (prepared by adding 1.48% Orvus ES Paste and 6% glycerol to the first diluent). The resuspended and cooled spermatozoa were packed into 0.5 mL straws, frozen in liquid nitrogen vapor at 4 cm above the surface, and then stored at −196 ◦ C until use. Semen samples were collected from six mature boars (four Duroc, one Berkshire, and one Landrace), packed in 0.5mL plastic straws at 1 × 109 spermatozoa/mL, then frozen and stored in liquid nitrogen until use. To thaw the frozen samples, one or two straws, depending on experiments, were immersed in water at 38 ◦ C for 30 s prior to use. The frozen-thawed spermatozoa were then diluted with mMS or with porcine fertilization medium (PFM; Research Institute for the Functional Peptides, Yamagata, Japan). This is commonly used as a liquid semen extender for storage in Japan. PFM is the medium used for IVF with porcine oocytes and spermatozoa, and contains theophylline, adenosine, and cysteine (Yoshioka et al., 2003). The composition of both extenders are shown in Table 1.
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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2.3. Assessment of sperm motility Sperm motility in each sample was assessed by computer-assisted semen analysis (CASA; CEROS Sperm Analyzer, Hamilton Thorne Biosciences, Beverly, MA, USA) at 0.5, 1.5, 3 and 6 h after thawing as described (Noguchi et al., 2015). Briefly, each sample (5 × 108 spermatozoa in a 0.5 mL straw) was diluted to 2 × 107 spermatozoa/mL in mMS or PFM. The CASA was setup with the following settings: 55 frames acquired, frame rate of 60 Hz, minimum contrast of 50, minimum cell size of seven pixels, straightness (STR) threshold of 80%, low average path velocity (VAP) cutoff of 7.2 m/s, medium VAP cutoff of 50 m/s, static head size of 0.66–5.50, magnification of 2.04 and a minimum of 200 sperm were observed for motility analysis. The different motility parameters evaluated were: percentage total motility (MOT, %); percentage progressive motility (PROG, %); VAP (m/s); straight-line velocity (VSL, m/s); curvilinear velocity (VCL, m/s); amplitude of lateral head displacement (ALH, m), beat cross-frequency (BCF, Hz); STR (VSL/VAP, %); and linearity (LIN = VSL/VCL, %). 2.4. Assessment of plasma and acrosomal membrane integrity The integrity of the plasma and acrosomal membranes was evaluated at 0.5, 1.5, 3 and 6 h after thawing following the procedure described by Noguchi et al. (2015). Briefly, each sample (5 × 108 spermatozoa in a 0.5 mL straw) after washing was diluted to 1 × 107 spermatozoa/mL in mMS or PFM. Spermatozoa were stained with final concentrations of 25 nM of SYBR-14 (Component A of the LIVE/DEAD Sperm Viability Kit, Molecular Probes, Eugene, OR, USA), 12 M of propidium iodide (PI) (Component B of the LIVE/DEAD Sperm Viability Kit) and 0.25 mg/mL of Alexa Fluor 647-conjugated peanut agglutinin (Molecular Probes). Stained sperm cells were analyzed subsequently in a flow cytometer (FACSAriaTM , Becton Dickinson, Franklin Lakes, NJ, USA). A total of 50,000 events with forward and side scatter properties of sperm cells were gated initially. Then, dots that were doubly negative for SYBR-14 and PI were further gated out as non-sperm particles. Spermatozoa were divided into four subpopulations: those with intact plasma and acrosomal membranes (IPIA; PI (−) and PNA (−)); those with intact plasma and damaged acrosomal membranes (IPDA; PI (−) and PNA (+)); those with damaged plasma and intact acrosomal membranes (DPIA; PI (+) and PNA (−)); and those with damaged plasma and acrosomal membrane (DPDA; PI (+) and PNA (+)). 2.5. Collection of uterine spermatozoa and PMNs Thirty sows were synchronized to estrus using injections of prostaglandin F2 alpha as 15 mg dinoprost (Panacelan Hi; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), equine chorionic gonadotropin 1000 IU (Serotropin; Aska Pharmaceutical Co., Tokyo, Japan) and human chorionic gonadotropin (hCG) 750 IU (Gonatropin; Aska Pharmaceutical Co.) as described by Yoshioka et al. (2003). They were inseminated once with frozen-thawed semen (5 × 108
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spermatozoa in a 0.5 mL straw) diluted in 5 mL of mMS (n = 15) or PFM (n = 15) 34 h after hCG injection using a deep intrauterine catheter (Takumi® ; length, 1.2 m; outer diameter, 3 mm; working duct, 0.5 mm; Fujihira Industry Co., Tokyo, Japan), and then 5.0 mL of the same extender was infused into the catheter to flush the semen. At 4 h after DIUI, the animals were anesthetized with an intravenously administered overdose of sodium pentobarbital (Somnopentyl, Kyoritsu, Tokyo, Japan), and the reproductive tracts were recovered at the Institute’s own abattoir and immediately transported to the laboratory. We confirmed that all sows had large follicles (over 6 mm in diameter) on the ovaries, with no evidence of ovulation. The oviducts and uterine horns on each side, and the uterine body were separated. Each oviduct was separated into three parts as previously described (Kunavongkrit et al., 2003): (1) the ampullary segment, comprises two-thirds of the oviduct next to the isthmus; (2) the isthmus segment, comprises one-third of the oviduct next to the UTJ; and (3) the UTJ segment incorporates 1 cm of the isthmus and 2 cm of the tip of the uterine horn. The method of sperm recovery was modified from that used in gilts by Kunavongkrit et al. (2003). Briefly, the ampulla, isthmus, UTJ segments, and uterine body were flushed twice with 5 mL of phosphate-buffered saline (PBS) through the lumen. The uterine horn was flushed twice with 25 mL of PBS. The sample from each part was centrifuged at 800 × g for 10 min at 4 ◦ C and then the supernatant was removed. Pellets recovered from oviduct segments (the ampulla, isthmus, and UTJ) were resuspended in 100 L PBS. Pellets from the uterine horns and body were resuspended in 5 and 1 mL PBS, respectively. The numbers of spermatozoa in the flushing from each sample were determined using a hemocytometer after adequate dilution with PBS. The number of PMNs in the uterine horn sample was also counted using a hemocytometer. After flushing the UTJ, the numbers of spermatozoa in the crypts were also counted as described by Tummaruk and Tienthai (2010). The UTJ was cut out at 5 mm from the isthmus and uterine horn sides. Then it was cut into four equal parts (5 mm long), immersed in a 10% neutral buffered formalin solution and embedded in a paraffin block. The embedded tissue sections were sectioned transversely to a thickness of 5 m. Every fifth serial section was mounted and stained with hematoxylin and eosin. The numbers of spermatozoa in defined sections were determined using 200–400 × light microscopy. 2.6. Artificial insemination, pregnancy diagnosis and reproductive parameters Twenty-two sows were synchronized to estrus as above. They were inseminated once with frozen-thawed semen (10 × 108 spermatozoa in two 0.5-mL straws) diluted in 5 mL of mMS (n = 11) or PFM (n = 11) 36 h after hCG injection using a deep intrauterine catheter as described above. Immediately after DIUI, 5 mL of the same extender was infused into the catheter to flush out residual semen into the uterus. Ovulation was detected by transrectal ultrasonography (SSD-900SE; Aloka, Tokyo, Japan) in all sows every 4 h from 30 h after hCG treatment as described by
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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Noguchi et al. (2010). Animals inseminated by DIUI were checked for estrus from day 17 after ovulation. If they did not exhibit a subsequent estrus until 25 days after ovulation, they were confirmed to be pregnant by ultrasound. All pregnant recipients were allowed to carry their litters to term and the pregnancy rate, farrowing rate, pregnancy period, total and live born litter size and piglet weight were recorded.
tested for normality of distribution using the Shapiro–Wilk test. Because the data were not distributed normally and variances were not homogeneous, the significance of differences between mMS and PFM treatments was determined using Mann–Whitney nonparametric U tests. The pregnancy and farrowing rates were examined using the Chi squared test. P < 0.05 was considered to be significant. 3. Results
2.7. Statistical analysis 3.1. Effects of semen extender on frozen-thawed sperm motility
Data on sperm motility characteristics as determined using CASA, numbers of spermatozoa and PMNs in reproductive tracts, and reproductive performance were analyzed using the general linear model procedures of the Statistical Analysis System (Ver. 9.4; SAS Institute Inc., Cary, NC, USA). The statistical model included the effects of semen extender, incubation time and interaction of semen extender × incubation time for experiments measuring sperm characteristics. When a significant effect was detected by analysis of variance, Tukey’s post-hoc analysis was used for pairwise comparisons. In the experiments of sperm distribution, migration of PMNs into the reproductive tract and the number of total and live-born piglets and mean piglet weight after DIUI, the data obtained were
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The parameters of sperm motility assessed using CASA are presented in Fig. 1. The MOT value at 3 h after thawing in the PFM treatment group was higher than in the mMS group (P < 0.05), but was not significantly different between treatments at 0.5, 1.5 and 6 h. The PROG, LIN, STR and VSL values in the PFM group were higher than those in the mMS group throughout the 6 h (P < 0.05). The VCL and ALH values in the PFM group were lower than those in the mMS group from 0.5 to 3 h after thawing (P < 0.05). The VAP value in the PFM group was lower at 0.5 h and higher at 3 h than in the mMS group (P < 0.05), respectively. The BCF at 1.5 h in the PFM group was lower than in the mMS group (P < 0.05).
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Fig. 1. Characteristics of frozen-thawed sperm motility when diluted in modified Modena solution (mMS) or porcine fertilization medium (PFM). Results are reported as percentages of total motility (MOT, %) and progressive motility (PROG, %), average path velocity (VAP, m/s), straight-line velocity (VSL, m/s), curvilinear velocity (VCL, m/s), amplitude of lateral head displacement (ALH, m), beat cross-frequency (BCF, Hz), straightness (STR = VSL/VAP, %) and linearity (LIN = VSL/VCL, %). Data are shown from 18 replicates (three for each of six boars). Different letters denote significant differences among times after thawing within each group (P < 0.05). Asterisks indicate significant differences between groups at each time (P < 0.05). Data are shown as the mean ± standard error of the mean (SEM; bars).
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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3.2. Effects of semen extender on plasma and acrosomal membrane integrity of frozen-thawed spermatozoa Data on the integrity of plasma and acrosomal membranes assessed by flow cytometry are shown in Fig. 2. The percentage of IPIA in the mMS extender was higher than that in PFM throughout the 6 h after thawing (P < 0.05). Higher percentages of spermatozoa with damaged acrosomal membranes (IPDA and DPDA) were observed in the PFM treatment group compared with the mMS group (P < 0.05). Although the percentage of sperm with damaged acrosomal membrane in the mMS group did not changed from 0.5 to 6 h after thawing, the percentage in PFM treatment was significantly (P < 0.05) higher at 3 and 6 h than at 0.5 h post-thawing. The percentages of DPIA at 3 and 6 h after thawing in the PFM group were higher than those in the mMS group (P < 0.05).
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(mMS group vs. PFM group = 8.8 ± 2.6 vs. 7.1 ± 1.7 × 103 spermatozoa/suspension) and in the crypts (mMS group vs. PFM group = 178.8 ± 74.4 vs. 229.6 ± 125.1 spermatozoa/mm) did not differ between the groups. The mean number of PMNs in the PFM group was significantly lower (P < 0.05) than in the mMS group in both the uterine body and horns (Fig. 3). The numbers of spermatozoa phagocytosed by PMNs in the uterus did not differ between the groups. However, the number of non-phagocytosed sperm (i.e. outside PMNs) in the uterus horn was significantly higher in PFM than in mMS groups. 3.4. Effects of semen extender on reproductive performance after DIUI The type of semen extender used did not have a significant effect on pregnancy and farrowing rates (Table 2). The numbers of total and live-born piglets and mean piglet weights also did not differ between the two treatments.
3.3. Effects of semen extender on sperm distribution in reproductive tract and PMNs migration into the uterine lumen
4. Discussion
No spermatozoa were observed in the ampulla in either treatment group. There were no differences in the number of spermatozoa between treatments in the isthmus. In the UTJ section, the number of spermatozoa in the flushing fluid
Treatment with PFM further enhanced the progressive motility parameters (PROG, VSL, STR and LIN) of frozen-thawed boar spermatozoa compared with mMS. Methylxanthines, such as caffeine and theophylline, inhibit
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Time after thawing (h) Fig. 2. Characteristics of sperm membranes diluted in mMS or PFM. Results are reported as four subpopulations: spermatozoa with both plasma and acrosomal membranes intact (IPIA; PI (−) and PNA (−)); spermatozoa with intact plasma and damaged acrosomal membranes (IPDA; PI (−) and PNA (+)); spermatozoa with damaged plasma and intact acrosomal membranes (DPIA; PI (+) and PNA (−)); and sperm with damaged plasma and acrosomal membranes (DPDA; PI (+) and PNA (+)). Data are from 18 replicates (three for each of six boars). Different letters denote significant differences among times after thawing within each group (P < 0.05). Asterisks indicate significant differences between groups at each time (P < 0.05). Data are shown as the mean ± SEM (bars).
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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Fig. 3. Distribution of polymorphonuclear leukocytes (PMNs), spermatozoa phagocytosed by PMNs and non-phagocytosed spermatozoa in uterine horns and uterine body after deep intrauterine insemination with frozen-thawed spermatozoa diluted in mMS or PFM. PMNs and spermatozoa were recovered from 15 sows in each group. Different letters denote significant differences between groups (P < 0.05). Data are shown as the mean ± SEM (bars).
Table 2 Reproductive performance after deep intrauterine insemination (DIUI) with frozen-thawed spermatozoa diluted in modified Modena solution (mMS) or porcine fertilization medium (PFM).
Number of animals Interval from DIUI to ovulation (h) Number of pregnancies (%) Number of farrowings (%) Pregnancy period (day) Number of total born piglets Number of live-born piglets Piglet weight (kg)
mMS
PFM
11 8.7 ± 1.6 3 (36) 3 (36) 114.8 ± 0.3 9.0 ± 3.0 7.5 ± 2.9 1.47 ± 0.06
11 7.3 ± 1.8 7 (64) 7 (64) 113.6 ± 0.6 8.4 ± 1.7 7.6 ± 1.7 1.40 ± 0.03
Data except for percentiles are expressed as the mean ± standard error of the mean (SEM).
phosphodiesterase activity (Fredholm, 1985). This in turn increases the intracellular concentrations of cAMP, a second messenger that enhances sperm motility and stimulates capacitation. Thus, these agents have been used for porcine IVF to enhance fertilization rates (Funahashi et al., 2000a,b; Yoshioka et al., 2003). Caffeine treatment enhanced progressive motility patterns in fresh (Yeste et al., 2008) and frozen-thawed (Yamaguchi et al., 2013) boar spermatozoa. The presence of theophylline in the PFM might have stimulated progressive motility in the frozenthawed boar spermatozoa in vitro, while it is still unclear whether theophylline influences characteristics of sperm motility in vivo. The addition of caffeine to mMS had no influence on the integrity of plasma or acrosomal membrane of
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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frozen-thawed boar spermatozoa in our previous study (Yamaguchi et al., 2013). However, the percentages of spermatozoa with both intact plasma and acrosomal membranes were significantly lower in PFM than in mMS throughout the 6-h study period in the present study. Intact sperm head membranes are a prerequisite for capacitation, subsequent hyperactivation, acrosome reaction and fertilization (Gadella et al., 2008). Although acrosome exocytosis in boar spermatozoa diluted with BTS supplemented with 1 mM caffeine were more frequent in mMS (not containing caffeine) after 0.5 and 1.5 h incubation (Yamaguchi et al., 2009), supplementation with 10 mM caffeine in mMS did not affect the percentages of IPIA sperm after 1.5 h incubation (Yamaguchi et al., 2013). Other authors have suggested that EDTA in this medium might have neutralized the stimulatory effect of caffeine on spontaneous acrosome reactions by scavenging the reactive oxygen species produced (Chi et al., 2008), and this hypothesis is consistent with our present results (mMS vs. PFM, 6.99 mM vs. 0 mM EDTA). Moreover, PFM includes some other factors, such as bicarbonate (Harrison and Gadella, 2005) and adenosine (Funahashi et al., 2000a; Yoshioka et al., 2003), which could elevate intracellular cAMP concentrations resulting in enhanced capacitation and/or acrosome reactions. From the present results and previous reports, it is possible that the membranes in frozen-thawed spermatozoa exposed to PFM were affected by components other than theophylline. Caffeine in semen extenders was able to regulate the migration of PMNs into the uterus because of suppression of interleukin (IL)-8 mRNA expression in the endometrium (Yamaguchi et al., 2009, 2013). IL-8 mRNA expression in endometrial tissue after porcine AI was correlated with the intrauterine recruitment of PMNs (Taylor et al., 2009). In our study, there were significant differences in the number of PMNs and in spermatozoa outside PMNs in the uterine horn between the mMS and PFM treatments. The results obtained from our study are in good agreement with previous reports using fresh (Matthijs et al., 2003) and BCC-diluted frozen-thawed spermatozoa (Yamaguchi et al., 2009, 2013) containing caffeine. Although the farrowing rates were not significantly different between the groups (mMS vs. PFM, 36% vs. 64%), we applied DIUI once with 1 × 109 frozen-thawed spermatozoa and our results were similar to previous reports that used AI twice with 2.5 × 109 frozen-thawed spermatozoa (mMS vs. BCC, 29% vs. 62%; Yamaguchi et al., 2009, 2013). These results indicate that the use of a modified semen extender for DIUI enables the breeder to reduce the numbers of spermatozoa inseminated per estrus and improve the efficiency of piglet production. When sows were inseminated deep into the uterine horn once with 1 × 109 frozen-thawed spermatozoa diluted with BTS, the pregnancy rate depended on the timing of insemination: 36.8–40.0% when inseminated at 4 or 8 h before ovulation (Wongtawan et al., 2006); and 79.6% when inseminated at 2–3 h before expected ovulation at 40 h after hCG injection (Roca et al., 2003). In the present study, sows were inseminated with 1 × 109 frozen-thawed spermatozoa at 4–8 h before expected ovulation at 34 h after hCG treatment. Thus, further experiments will be required for defining the optimal timing of DIUI using PFM.
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5. Conclusion In conclusion, our results demonstrate that PFM can be used as an extender of frozen-thawed boar semen. PFM enhanced the progressive motility of boar sperm, while it increases sperm membrane damage compared with mMS. Our results also indicate that PFM suppresses PMNs migration into the uterine lumen. Conflicts of interest The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Acknowledgements This research was supported in part by a grant for Research and Development Projects Promoting New Policies in Agriculture, Forestry and Fisheries (No. 21011) from the Ministry of Agriculture, Forestry and Fisheries of Japan and a Grant-in-Aid for Scientific Research B (No. 25292184) from the Japan Society for the Promotion of Science (JSPS). References Chi, H.J., Kim, J.H., Ryu, C.S., Lee, J.Y., Park, J.S., Chung, D.Y., Choi, S.Y., Kim, M.H., Chun, E.K., Roh, S.I., 2008. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum. Reprod. 23, 1023–1028. Fredholm, B.B., 1985. On the mechanism of action of theophylline and caffeine. Acta Med. Scand. 217, 149–153. Funahashi, H., Asano, A., Fujiwara, T., Nagai, T., Niwa, K., Fraser, L.R., 2000a. Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro. Mol. Reprod. Dev. 55, 117–124. Funahashi, H., Fujiwara, T., Nagai, T., 2000b. Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes. Biol. Reprod. 63, 1157–1163. Funahashi, H., Nagai, T., 2001. Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine. Mol. Reprod. Dev. 58, 424–431. Gadella, B.M., Tsai, P.-S., Boerke, A., Brewis, I.A., 2008. Sperm head membrane reorganisation during capacitation. Int. J. Dev. Biol. 52, 473–480. Harrison, R.A.P., Gadella, B.M., 2005. Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 63, 342–351. Krueger, C., Rath, D., Johnson, L.A., 1999. Low dose insemination synchronized gilts. Theriogenology 52, 1363–1373. Kunavongkrit, A., Sang-Gasanee, K., Phumratanaprapin, C., Tantasuparuk, W., Einarsson, S., 2003. A study on the number of recovered spermatozoa in the uterine horns and oviducts of gilts, after fractionated or non-fractionated insemination. J. Vet. Med. Sci. 65, 63–67. Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A., Parrilla, I.J., Vazquez, L., Day, B.N., 2001. Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows. Reproduction 122, 289–296. Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A., Parrilla, I.J., Vazquez, L., Day, B.N., 2002. Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows. Reproduction 123, 163–170. Matthijs, A., Engel, B., Woelders, H., 2003. Neutrophil recruitment and phagocytosis of boar spermatozoa after artificial insemination of sows, and the effects of inseminate volume, sperm dose and specific additives in the extender. Reproduction 125, 357–367. Noguchi, M., Yoshioka, K., Itoh, S., Suzuki, C., Arai, S., Wada, Y., Hasegawa, Y., Kaneko, H., 2010. Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow. Reproduction 139, 153–161. Noguchi, M., Yoshioka, K., Hikono, H., Iwagami, G., Suzuki, C., Kikuchi, K., 2015. Centrifugation on Percoll density gradient enhances motility,
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membrane integrity and in vitro fertilizing ability of frozen-thawed boar sperm. Zygote 23, 68–75. Penã, F.J., Johannisson, A., Wallgrena, M., Rodríguez-Martínez, H., 2003. Assessment of fresh and frozen-thawed boar semen using an Annexin-V assay: a new method of evaluating sperm membrane integrity. Theriogenology 60, 677–689. Roca, J., Carvajal, G., Lucas, X., Vázquez, J.M., Martínez, E.A., 2003. Fertility of weaned sows after deep intrauterine insemination with a reduced number of frozen-thawed spermatozoa. Theriogenology 60, 77–87. Roca, J., Hernández, M., Carvajal, G., Vázquez, J.M., Martínez, E.A., 2006a. Factors influencing boar sperm cryosurvival. J. Anim. Sci. 84, 2692–2699. Roca, J., Vázquez, J.M., Gil, M.A., Cuello, C., Parrilla, I., Martínez, E.A., 2006b. Challenges in pig artificial insemination. Reprod. Dom. Anim. Suppl. 41, 43–53. Steverink, D.W.B., Soede, N.M., Bouwman, E.G., Kemp, B., 1998. Semen backflow after insemination and its effect on fertilisation results in sows. Anim. Reprod. Sci. 54, 109–119. Taylor, U., Zerbe, H., Seyfert, H.-M., Rath, D., Baulain, U., Langnerd, K.F.A., Schuberth, H.-J., 2009. Porcine spermatozoa inhibit post-breeding cytokine induction in uterine epithelial cells in vivo. Anim. Reprod. Sci. 115, 279–289. Tummaruk, P., Tienthai, P., 2010. Number of spermatozoa in the crypts of the sperm reservoir at about 24 h after a low-dose intrauterine and
deep intrauterine insemination in sows. Reprod. Domest. Anim. 45, 208–213. Wongtawan, T., Saravia, F., Wallgren, M., Caballero, I., Rodríguez-Martínez, H., 2006. Fertility after deep intra-uterine artificial insemination of concentrated low-volume boar semen doses. Theriogenology 65, 773–787. Yamaguchi, S., Funahashi, H., Murakami, T., 2009. Improved fertility in gilts and sows after artificial insemination of frozen-thawed boar semen by supplementation of semen extender with caffeine and CaCl2 . J. Reprod. Dev. 55, 645–649. Yamaguchi, S., Suzuki, C., Noguchi, M., Kasa, S., Mori, M., Isozaki, Y., Ueda, S., Funahashi, H., Kikuchi, K., Nagai, T., Yoshioka, K., 2013. Effects of caffeine on sperm characteristics after thawing and inflammatory response in the uterus after artificial insemination with frozen-thawed boar semen. Theriogenology 79, 87–93. Yeste, M., Briz, M., Pinart, E., Sancho, S., Garcia-Gil, N., Badia, E., Bassols, J., Pruneda, A., Bussalleu, E., Casas, I., Bonet, S., 2008. Hyaluronic acid delays boar sperm capacitation after 3 days of storage at 15 ◦ C. Anim. Reprod. Sci. 109, 236–250. Yoshioka, K., Suzuki, C., Itoh, S., Kikuchi, K., Iwamura, S., Rodríguez-Martínez, H., 2003. Production of piglets derived from in vitro-produced blastocysts fertilized and cultured in chemically defined media: effects of theophylline, adenosine, and cysteine during in vitro fertilization. Biol. Reprod. 69, 2092–2099.
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Animal Reproduction Science journal homepage: www.elsevier.com/locate/anireprosci
Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows Michiko Noguchi a,1 , Koji Yoshioka a,∗ , Hirokazu Hikono a , Chie Suzuki a , Kazuhiro Kikuchi b a b
National Institute of Animal Health, Tsukuba, Ibaraki, Japan National Institute of Agrobiological Sciences, Tsukuba, Ibaraki, Japan
a r t i c l e
i n f o
Article history: Received 13 June 2015 Received in revised form 29 August 2015 Accepted 2 November 2015 Available online xxx Keywords: Adenosine Deep intrauterine insemination Frozen-thawed semen Pig Theophylline
a b s t r a c t We compared the effects of extenders of frozen-thawed semen on post-thaw sperm characteristics and the distribution of frozen-thawed spermatozoa in the female genital tract after fixed-timed deep intrauterine insemination (DIUI) in sows. Frozen semen samples were thawed and diluted in either modified Modena solution (mMS) or porcine fertilization medium (PFM) containing theophylline, adenosine and cysteine. Sperm quality, assessed in vitro based on motility using a computer-assisted sperm analyzer and the integrity of the plasma and acrosomal membranes using flow cytometry, was evaluated at 0.5, 1.5, 3 and 6 h after thawing. Progressive motility and the percentage of spermatozoa with damaged acrosomal membranes in PFM were significantly better than in mMS throughout the 6 h. Sows with estrus synchronized using prostaglandin F2 alpha, equine chorionic gonadotropin and human chorionic gonadotropin (hCG) were inseminated once with mMS- or PFM-diluted 5 × 108 frozen-thawed spermatozoa by DIUI at 34 h after the hCG injection. At 4 h after DIUI, reproductive tracts were recovered from 30 sows. There were significantly fewer polymorphonuclear leukocytes (PMNs) and more spermatozoa outside PMNs in the uterine horn after PFM treatment than with mMS. When 22 sows were administered DIUI with 10 × 108 frozen-thawed spermatozoa at 36 h after hCG, the pregnancy rates did not differ significantly between the mMS- (36%) and PFM- (64%) treated groups. Thus, PFM enhanced progressive sperm motility but increased sperm membrane damage compared with mMS; it also suppressed the migration of PMNs into the uterine lumen. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Artificial insemination (AI) techniques for pig breeding have been focused on reducing the number of
∗ Corresponding author at: National Institute of Animal Health, Kannondai 3-1-5, Tsukuba, Ibaraki 305-0856, Japan. E-mail address: [email protected] (K. Yoshioka). 1 Present address: Laboratory of Theriogenology, Department of Veterinary Medicine, Azabu University, Sagamihara, Kanagawa, Japan.
spermatozoa per dose and/or determining optimal AI timing during estrus while maintaining good reproductive performance (reviewed by Roca et al., 2006b). Successful fertilization by AI in pigs depends on the number of spermatozoa in the uterotubal junction (UTJ) around the time of ovulation (Krueger et al., 1999). Approximately 20–45% of the spermatozoa inseminated into the cervix are lost as they flow back out of the reproductive tract (Steverink et al., 1998; Matthijs et al., 2003). The technique of deep intrauterine insemination (DIUI) can diminish the backflow of semen from the genital tract (Martinez et al.,
http://dx.doi.org/10.1016/j.anireprosci.2015.11.006 0378-4320/© 2015 Elsevier B.V. All rights reserved.
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2001, 2002). Furthermore, farrowing rates and litter sizes in sows after DIUI with 1000 × 106 frozen-thawed spermatozoa are equivalent to those of sows inseminated with 6000 × 106 frozen-thawed spermatozoa into the cervix (Roca et al., 2003). Therefore, despite the poor condition of frozen-thawed spermatozoa compared with those of fresh semen (Penã et al., 2003; Roca et al., 2006a), they have been used in DIUI as an efficient tool for piglet production (Roca et al., 2003; Wongtawan et al., 2006). When sows are inseminated into the cervix semen backflow and sperm cell phagocytosis by polymorphonuclear leukocytes (PMNs) leads to loss of spermatozoa in the uterus. It has been reported that migration of PMNs into the uterus and subsequent sperm phagocytosis depends on the composition of the semen extender used in pigs (Matthijs et al., 2003; Yamaguchi et al., 2009). Methylxanthines, especially caffeine, stimulate both capacitation and spontaneous acrosome reaction of boar spermatozoa in vitro (Funahashi et al., 2000a; Funahashi and Nagai, 2001). In vivo experiments showed that caffeine could suppress the intrauterine migration of PMNs when pigs were artificially inseminated with fresh or frozen-thawed semen diluted with Beltsville thawing solution (BTS) supplemented with caffeine and calcium chloride (CaCl2 ) (BCC) (Matthijs et al., 2003; Yamaguchi et al., 2009). Because of this inhibition of PMN migration with caffeine, the numbers of spermatozoa in the oviducts, and the pregnancy and farrowing rates produced by frozen-thawed semen diluted with BCC were higher than those of semen diluted with Modena solution, which does not contain caffeine (Yamaguchi et al., 2009). Moreover, the addition of caffeine to the modified Modena solution for thawing enhanced the motion quality parameters of frozen-thawed boar sperm with no influence on the integrity of plasma or acrosomal membranes (Yamaguchi et al., 2013). These results indicate that caffeine supplementation of semen extenders can enhance sperm transportation into the UTJ and improve reproductive performance in vivo. Adenosine, which is present in the female reproductive tract, stimulates capacitation and inhibits spontaneous acrosome reactions in boar spermatozoa (Funahashi et al., 2000a,b; Funahashi and Nagai, 2001). Adenosine supplementation of in vitro fertilization (IVF) medium can produce better sperm penetration rates than methylxanthine supplementation (Funahashi et al., 2000b; Funahashi and Nagai, 2001; Yoshioka et al., 2003). These reports suggest that supplementation with adenosine in semen extenders might enhance reproductive performance after DIUI with spermatozoa with low quality and/or in a small volume. The objectives of this study were to determine the effects of IVF medium supplemented with methylxanthine and adenosine as a semen extender on (1) sperm characteristics, (2) distribution of spermatozoa in the female genital tract and (3) reproductive performance in sows after DIUI. 2. Materials and methods 2.1. Animals All animal-related procedures followed in this study were approved by the Institutional Care and Use
Table 1 Components of the semen extenders used. Component
mMS
PFM
NaCl (mM) KCl (mM) KH2 PO4 (mM) MgSO4 ·7H2 O (mM) NaHCO3 (mM) Glucose (mM) Na-pyruvate (mM) Ca-(lactate)2 ·5H2 O (mM) Theophylline (mM) Adenosine (M) l-cysteine (M) Gentamicin (g/mL) PVA (mg/mL) Na-citrate·2H2 O (mM) Citric acid (mM) EDTA·2Na (mM) Tris (mM)
– – – – 11.90 152.61 – – – – – 10 – 23.46 15.10 6.99 46.66
108.0 10.0 0.35 0.40 25.07 1.00 0.2 4.0 2.5 1.0 0.25 10 3.0 – – – –
mMS, modified Modena solution; PFM, porcine fertilization medium.
Committee for Laboratory Animals of the National Institute of Animal Health in Japan (Protocol No. 11-003). Animals were kept in individual crates with water available ad libitum and received 2.2 kg of a commercial diet once daily. Estrus detection in sows was performed twice daily using a mature boar. Estrus was defined as the period during which sows exhibited a standing response for the boar. 2.2. Preparation of frozen-thawed boar spermatozoa and semen extenders The method of semen cryopreservation was essentially the same as that described by Yoshioka et al. (2003) with minor modifications. Briefly, the collected semen was diluted 1:2 with Modena solution supplemented with 10g/mL gentamycin (mMS; Table 1) and kept overnight at 15 ◦ C. After centrifugation at 800 × g for 10 min, the precipitated spermatozoa were resuspended at a density of 2 × 109 cells/mL in the first diluent (0.3 M lactose solution containing 20% egg yolk, 75 mg/mL amikacin, 25 mg/mL dibekacin and 2,500,000 IU/mL potassium penicillin G) and then cooled to 5 ◦ C for 2–3 h. The samples were then further diluted 1:1 with the second diluent (prepared by adding 1.48% Orvus ES Paste and 6% glycerol to the first diluent). The resuspended and cooled spermatozoa were packed into 0.5 mL straws, frozen in liquid nitrogen vapor at 4 cm above the surface, and then stored at −196 ◦ C until use. Semen samples were collected from six mature boars (four Duroc, one Berkshire, and one Landrace), packed in 0.5mL plastic straws at 1 × 109 spermatozoa/mL, then frozen and stored in liquid nitrogen until use. To thaw the frozen samples, one or two straws, depending on experiments, were immersed in water at 38 ◦ C for 30 s prior to use. The frozen-thawed spermatozoa were then diluted with mMS or with porcine fertilization medium (PFM; Research Institute for the Functional Peptides, Yamagata, Japan). This is commonly used as a liquid semen extender for storage in Japan. PFM is the medium used for IVF with porcine oocytes and spermatozoa, and contains theophylline, adenosine, and cysteine (Yoshioka et al., 2003). The composition of both extenders are shown in Table 1.
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2.3. Assessment of sperm motility Sperm motility in each sample was assessed by computer-assisted semen analysis (CASA; CEROS Sperm Analyzer, Hamilton Thorne Biosciences, Beverly, MA, USA) at 0.5, 1.5, 3 and 6 h after thawing as described (Noguchi et al., 2015). Briefly, each sample (5 × 108 spermatozoa in a 0.5 mL straw) was diluted to 2 × 107 spermatozoa/mL in mMS or PFM. The CASA was setup with the following settings: 55 frames acquired, frame rate of 60 Hz, minimum contrast of 50, minimum cell size of seven pixels, straightness (STR) threshold of 80%, low average path velocity (VAP) cutoff of 7.2 m/s, medium VAP cutoff of 50 m/s, static head size of 0.66–5.50, magnification of 2.04 and a minimum of 200 sperm were observed for motility analysis. The different motility parameters evaluated were: percentage total motility (MOT, %); percentage progressive motility (PROG, %); VAP (m/s); straight-line velocity (VSL, m/s); curvilinear velocity (VCL, m/s); amplitude of lateral head displacement (ALH, m), beat cross-frequency (BCF, Hz); STR (VSL/VAP, %); and linearity (LIN = VSL/VCL, %). 2.4. Assessment of plasma and acrosomal membrane integrity The integrity of the plasma and acrosomal membranes was evaluated at 0.5, 1.5, 3 and 6 h after thawing following the procedure described by Noguchi et al. (2015). Briefly, each sample (5 × 108 spermatozoa in a 0.5 mL straw) after washing was diluted to 1 × 107 spermatozoa/mL in mMS or PFM. Spermatozoa were stained with final concentrations of 25 nM of SYBR-14 (Component A of the LIVE/DEAD Sperm Viability Kit, Molecular Probes, Eugene, OR, USA), 12 M of propidium iodide (PI) (Component B of the LIVE/DEAD Sperm Viability Kit) and 0.25 mg/mL of Alexa Fluor 647-conjugated peanut agglutinin (Molecular Probes). Stained sperm cells were analyzed subsequently in a flow cytometer (FACSAriaTM , Becton Dickinson, Franklin Lakes, NJ, USA). A total of 50,000 events with forward and side scatter properties of sperm cells were gated initially. Then, dots that were doubly negative for SYBR-14 and PI were further gated out as non-sperm particles. Spermatozoa were divided into four subpopulations: those with intact plasma and acrosomal membranes (IPIA; PI (−) and PNA (−)); those with intact plasma and damaged acrosomal membranes (IPDA; PI (−) and PNA (+)); those with damaged plasma and intact acrosomal membranes (DPIA; PI (+) and PNA (−)); and those with damaged plasma and acrosomal membrane (DPDA; PI (+) and PNA (+)). 2.5. Collection of uterine spermatozoa and PMNs Thirty sows were synchronized to estrus using injections of prostaglandin F2 alpha as 15 mg dinoprost (Panacelan Hi; Meiji Seika Pharma Co. Ltd., Tokyo, Japan), equine chorionic gonadotropin 1000 IU (Serotropin; Aska Pharmaceutical Co., Tokyo, Japan) and human chorionic gonadotropin (hCG) 750 IU (Gonatropin; Aska Pharmaceutical Co.) as described by Yoshioka et al. (2003). They were inseminated once with frozen-thawed semen (5 × 108
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spermatozoa in a 0.5 mL straw) diluted in 5 mL of mMS (n = 15) or PFM (n = 15) 34 h after hCG injection using a deep intrauterine catheter (Takumi® ; length, 1.2 m; outer diameter, 3 mm; working duct, 0.5 mm; Fujihira Industry Co., Tokyo, Japan), and then 5.0 mL of the same extender was infused into the catheter to flush the semen. At 4 h after DIUI, the animals were anesthetized with an intravenously administered overdose of sodium pentobarbital (Somnopentyl, Kyoritsu, Tokyo, Japan), and the reproductive tracts were recovered at the Institute’s own abattoir and immediately transported to the laboratory. We confirmed that all sows had large follicles (over 6 mm in diameter) on the ovaries, with no evidence of ovulation. The oviducts and uterine horns on each side, and the uterine body were separated. Each oviduct was separated into three parts as previously described (Kunavongkrit et al., 2003): (1) the ampullary segment, comprises two-thirds of the oviduct next to the isthmus; (2) the isthmus segment, comprises one-third of the oviduct next to the UTJ; and (3) the UTJ segment incorporates 1 cm of the isthmus and 2 cm of the tip of the uterine horn. The method of sperm recovery was modified from that used in gilts by Kunavongkrit et al. (2003). Briefly, the ampulla, isthmus, UTJ segments, and uterine body were flushed twice with 5 mL of phosphate-buffered saline (PBS) through the lumen. The uterine horn was flushed twice with 25 mL of PBS. The sample from each part was centrifuged at 800 × g for 10 min at 4 ◦ C and then the supernatant was removed. Pellets recovered from oviduct segments (the ampulla, isthmus, and UTJ) were resuspended in 100 L PBS. Pellets from the uterine horns and body were resuspended in 5 and 1 mL PBS, respectively. The numbers of spermatozoa in the flushing from each sample were determined using a hemocytometer after adequate dilution with PBS. The number of PMNs in the uterine horn sample was also counted using a hemocytometer. After flushing the UTJ, the numbers of spermatozoa in the crypts were also counted as described by Tummaruk and Tienthai (2010). The UTJ was cut out at 5 mm from the isthmus and uterine horn sides. Then it was cut into four equal parts (5 mm long), immersed in a 10% neutral buffered formalin solution and embedded in a paraffin block. The embedded tissue sections were sectioned transversely to a thickness of 5 m. Every fifth serial section was mounted and stained with hematoxylin and eosin. The numbers of spermatozoa in defined sections were determined using 200–400 × light microscopy. 2.6. Artificial insemination, pregnancy diagnosis and reproductive parameters Twenty-two sows were synchronized to estrus as above. They were inseminated once with frozen-thawed semen (10 × 108 spermatozoa in two 0.5-mL straws) diluted in 5 mL of mMS (n = 11) or PFM (n = 11) 36 h after hCG injection using a deep intrauterine catheter as described above. Immediately after DIUI, 5 mL of the same extender was infused into the catheter to flush out residual semen into the uterus. Ovulation was detected by transrectal ultrasonography (SSD-900SE; Aloka, Tokyo, Japan) in all sows every 4 h from 30 h after hCG treatment as described by
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Noguchi et al. (2010). Animals inseminated by DIUI were checked for estrus from day 17 after ovulation. If they did not exhibit a subsequent estrus until 25 days after ovulation, they were confirmed to be pregnant by ultrasound. All pregnant recipients were allowed to carry their litters to term and the pregnancy rate, farrowing rate, pregnancy period, total and live born litter size and piglet weight were recorded.
tested for normality of distribution using the Shapiro–Wilk test. Because the data were not distributed normally and variances were not homogeneous, the significance of differences between mMS and PFM treatments was determined using Mann–Whitney nonparametric U tests. The pregnancy and farrowing rates were examined using the Chi squared test. P < 0.05 was considered to be significant. 3. Results
2.7. Statistical analysis 3.1. Effects of semen extender on frozen-thawed sperm motility
Data on sperm motility characteristics as determined using CASA, numbers of spermatozoa and PMNs in reproductive tracts, and reproductive performance were analyzed using the general linear model procedures of the Statistical Analysis System (Ver. 9.4; SAS Institute Inc., Cary, NC, USA). The statistical model included the effects of semen extender, incubation time and interaction of semen extender × incubation time for experiments measuring sperm characteristics. When a significant effect was detected by analysis of variance, Tukey’s post-hoc analysis was used for pairwise comparisons. In the experiments of sperm distribution, migration of PMNs into the reproductive tract and the number of total and live-born piglets and mean piglet weight after DIUI, the data obtained were
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a STR (%)
38
b 1
0
a
ab 0
a
d
c
30
6
b*
110
6
50
*
70 0
a*
a
b*
80
10
a
6
130 b*
b
60
40
ALH (μm)
a* ab*
50 VSL (μm/s)
b* 3
mMS PFM
a*
70
a*
140 a*
60
BCF (Hz)
80
LIN (%)
MOT (%)
70
40 a* a* 35 30 25 20 15 a 10 b 5 0 0 1 2
VAP (μm/s)
a
80
PROG (%)
90
The parameters of sperm motility assessed using CASA are presented in Fig. 1. The MOT value at 3 h after thawing in the PFM treatment group was higher than in the mMS group (P < 0.05), but was not significantly different between treatments at 0.5, 1.5 and 6 h. The PROG, LIN, STR and VSL values in the PFM group were higher than those in the mMS group throughout the 6 h (P < 0.05). The VCL and ALH values in the PFM group were lower than those in the mMS group from 0.5 to 3 h after thawing (P < 0.05). The VAP value in the PFM group was lower at 0.5 h and higher at 3 h than in the mMS group (P < 0.05), respectively. The BCF at 1.5 h in the PFM group was lower than in the mMS group (P < 0.05).
2
3
4
5
6
20 0
1 2 3 4 5 6 Time after thawing (h)
0
1
2
3
4
5
6
Fig. 1. Characteristics of frozen-thawed sperm motility when diluted in modified Modena solution (mMS) or porcine fertilization medium (PFM). Results are reported as percentages of total motility (MOT, %) and progressive motility (PROG, %), average path velocity (VAP, m/s), straight-line velocity (VSL, m/s), curvilinear velocity (VCL, m/s), amplitude of lateral head displacement (ALH, m), beat cross-frequency (BCF, Hz), straightness (STR = VSL/VAP, %) and linearity (LIN = VSL/VCL, %). Data are shown from 18 replicates (three for each of six boars). Different letters denote significant differences among times after thawing within each group (P < 0.05). Asterisks indicate significant differences between groups at each time (P < 0.05). Data are shown as the mean ± standard error of the mean (SEM; bars).
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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3.2. Effects of semen extender on plasma and acrosomal membrane integrity of frozen-thawed spermatozoa Data on the integrity of plasma and acrosomal membranes assessed by flow cytometry are shown in Fig. 2. The percentage of IPIA in the mMS extender was higher than that in PFM throughout the 6 h after thawing (P < 0.05). Higher percentages of spermatozoa with damaged acrosomal membranes (IPDA and DPDA) were observed in the PFM treatment group compared with the mMS group (P < 0.05). Although the percentage of sperm with damaged acrosomal membrane in the mMS group did not changed from 0.5 to 6 h after thawing, the percentage in PFM treatment was significantly (P < 0.05) higher at 3 and 6 h than at 0.5 h post-thawing. The percentages of DPIA at 3 and 6 h after thawing in the PFM group were higher than those in the mMS group (P < 0.05).
5
(mMS group vs. PFM group = 8.8 ± 2.6 vs. 7.1 ± 1.7 × 103 spermatozoa/suspension) and in the crypts (mMS group vs. PFM group = 178.8 ± 74.4 vs. 229.6 ± 125.1 spermatozoa/mm) did not differ between the groups. The mean number of PMNs in the PFM group was significantly lower (P < 0.05) than in the mMS group in both the uterine body and horns (Fig. 3). The numbers of spermatozoa phagocytosed by PMNs in the uterus did not differ between the groups. However, the number of non-phagocytosed sperm (i.e. outside PMNs) in the uterus horn was significantly higher in PFM than in mMS groups. 3.4. Effects of semen extender on reproductive performance after DIUI The type of semen extender used did not have a significant effect on pregnancy and farrowing rates (Table 2). The numbers of total and live-born piglets and mean piglet weights also did not differ between the two treatments.
3.3. Effects of semen extender on sperm distribution in reproductive tract and PMNs migration into the uterine lumen
4. Discussion
No spermatozoa were observed in the ampulla in either treatment group. There were no differences in the number of spermatozoa between treatments in the isthmus. In the UTJ section, the number of spermatozoa in the flushing fluid
Treatment with PFM further enhanced the progressive motility parameters (PROG, VSL, STR and LIN) of frozen-thawed boar spermatozoa compared with mMS. Methylxanthines, such as caffeine and theophylline, inhibit
50
a*
40
b 35 a
30
PFM
a*
30
b* c*
25
d*
20 10
b
20 0
1
2
3
5 4
5
6
0
1
2
3
4
5
a*
a*
60
6 a*
20
70 a*
15 b*
50 40
a
a
a
30
IPDA (%)
IPIA (%)
40
15
a
ab
25
mMS
35
b*
b
DPDA (%)
DPIA (%)
45
45
b*
10 5
20
bc
c
b 0
10 0
1
2
3
4
5
6
0
1
2
3
4
5
6
Time after thawing (h) Fig. 2. Characteristics of sperm membranes diluted in mMS or PFM. Results are reported as four subpopulations: spermatozoa with both plasma and acrosomal membranes intact (IPIA; PI (−) and PNA (−)); spermatozoa with intact plasma and damaged acrosomal membranes (IPDA; PI (−) and PNA (+)); spermatozoa with damaged plasma and intact acrosomal membranes (DPIA; PI (+) and PNA (−)); and sperm with damaged plasma and acrosomal membranes (DPDA; PI (+) and PNA (+)). Data are from 18 replicates (three for each of six boars). Different letters denote significant differences among times after thawing within each group (P < 0.05). Asterisks indicate significant differences between groups at each time (P < 0.05). Data are shown as the mean ± SEM (bars).
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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Uterine horns
No. of PMNs (×108)
16
Uterine body 0.8
a
14
a
0.6
12 10
b
8
b
0.4
6 4
0.2
2 0 PFM
mMS
No. of phagocytosed sperm by PMNs (×105)
14
mMS
PFM
mMS
PFM
mMS
PFM
1.0
12
0.8
10 8
0.6
6
0.4
4 0.2
2 0 mMS
PFM
a
30 No. of sperm outside PMNs (×105)
0
25
0
2.5 2.0
20
1.5
15
b
1.0
10
0.5
5 0
0 PFM
mMS
Fig. 3. Distribution of polymorphonuclear leukocytes (PMNs), spermatozoa phagocytosed by PMNs and non-phagocytosed spermatozoa in uterine horns and uterine body after deep intrauterine insemination with frozen-thawed spermatozoa diluted in mMS or PFM. PMNs and spermatozoa were recovered from 15 sows in each group. Different letters denote significant differences between groups (P < 0.05). Data are shown as the mean ± SEM (bars).
Table 2 Reproductive performance after deep intrauterine insemination (DIUI) with frozen-thawed spermatozoa diluted in modified Modena solution (mMS) or porcine fertilization medium (PFM).
Number of animals Interval from DIUI to ovulation (h) Number of pregnancies (%) Number of farrowings (%) Pregnancy period (day) Number of total born piglets Number of live-born piglets Piglet weight (kg)
mMS
PFM
11 8.7 ± 1.6 3 (36) 3 (36) 114.8 ± 0.3 9.0 ± 3.0 7.5 ± 2.9 1.47 ± 0.06
11 7.3 ± 1.8 7 (64) 7 (64) 113.6 ± 0.6 8.4 ± 1.7 7.6 ± 1.7 1.40 ± 0.03
Data except for percentiles are expressed as the mean ± standard error of the mean (SEM).
phosphodiesterase activity (Fredholm, 1985). This in turn increases the intracellular concentrations of cAMP, a second messenger that enhances sperm motility and stimulates capacitation. Thus, these agents have been used for porcine IVF to enhance fertilization rates (Funahashi et al., 2000a,b; Yoshioka et al., 2003). Caffeine treatment enhanced progressive motility patterns in fresh (Yeste et al., 2008) and frozen-thawed (Yamaguchi et al., 2013) boar spermatozoa. The presence of theophylline in the PFM might have stimulated progressive motility in the frozenthawed boar spermatozoa in vitro, while it is still unclear whether theophylline influences characteristics of sperm motility in vivo. The addition of caffeine to mMS had no influence on the integrity of plasma or acrosomal membrane of
Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006
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frozen-thawed boar spermatozoa in our previous study (Yamaguchi et al., 2013). However, the percentages of spermatozoa with both intact plasma and acrosomal membranes were significantly lower in PFM than in mMS throughout the 6-h study period in the present study. Intact sperm head membranes are a prerequisite for capacitation, subsequent hyperactivation, acrosome reaction and fertilization (Gadella et al., 2008). Although acrosome exocytosis in boar spermatozoa diluted with BTS supplemented with 1 mM caffeine were more frequent in mMS (not containing caffeine) after 0.5 and 1.5 h incubation (Yamaguchi et al., 2009), supplementation with 10 mM caffeine in mMS did not affect the percentages of IPIA sperm after 1.5 h incubation (Yamaguchi et al., 2013). Other authors have suggested that EDTA in this medium might have neutralized the stimulatory effect of caffeine on spontaneous acrosome reactions by scavenging the reactive oxygen species produced (Chi et al., 2008), and this hypothesis is consistent with our present results (mMS vs. PFM, 6.99 mM vs. 0 mM EDTA). Moreover, PFM includes some other factors, such as bicarbonate (Harrison and Gadella, 2005) and adenosine (Funahashi et al., 2000a; Yoshioka et al., 2003), which could elevate intracellular cAMP concentrations resulting in enhanced capacitation and/or acrosome reactions. From the present results and previous reports, it is possible that the membranes in frozen-thawed spermatozoa exposed to PFM were affected by components other than theophylline. Caffeine in semen extenders was able to regulate the migration of PMNs into the uterus because of suppression of interleukin (IL)-8 mRNA expression in the endometrium (Yamaguchi et al., 2009, 2013). IL-8 mRNA expression in endometrial tissue after porcine AI was correlated with the intrauterine recruitment of PMNs (Taylor et al., 2009). In our study, there were significant differences in the number of PMNs and in spermatozoa outside PMNs in the uterine horn between the mMS and PFM treatments. The results obtained from our study are in good agreement with previous reports using fresh (Matthijs et al., 2003) and BCC-diluted frozen-thawed spermatozoa (Yamaguchi et al., 2009, 2013) containing caffeine. Although the farrowing rates were not significantly different between the groups (mMS vs. PFM, 36% vs. 64%), we applied DIUI once with 1 × 109 frozen-thawed spermatozoa and our results were similar to previous reports that used AI twice with 2.5 × 109 frozen-thawed spermatozoa (mMS vs. BCC, 29% vs. 62%; Yamaguchi et al., 2009, 2013). These results indicate that the use of a modified semen extender for DIUI enables the breeder to reduce the numbers of spermatozoa inseminated per estrus and improve the efficiency of piglet production. When sows were inseminated deep into the uterine horn once with 1 × 109 frozen-thawed spermatozoa diluted with BTS, the pregnancy rate depended on the timing of insemination: 36.8–40.0% when inseminated at 4 or 8 h before ovulation (Wongtawan et al., 2006); and 79.6% when inseminated at 2–3 h before expected ovulation at 40 h after hCG injection (Roca et al., 2003). In the present study, sows were inseminated with 1 × 109 frozen-thawed spermatozoa at 4–8 h before expected ovulation at 34 h after hCG treatment. Thus, further experiments will be required for defining the optimal timing of DIUI using PFM.
7
5. Conclusion In conclusion, our results demonstrate that PFM can be used as an extender of frozen-thawed boar semen. PFM enhanced the progressive motility of boar sperm, while it increases sperm membrane damage compared with mMS. Our results also indicate that PFM suppresses PMNs migration into the uterine lumen. Conflicts of interest The authors declare that there is no conflict of interest that would prejudice the impartiality of this scientific work. Acknowledgements This research was supported in part by a grant for Research and Development Projects Promoting New Policies in Agriculture, Forestry and Fisheries (No. 21011) from the Ministry of Agriculture, Forestry and Fisheries of Japan and a Grant-in-Aid for Scientific Research B (No. 25292184) from the Japan Society for the Promotion of Science (JSPS). References Chi, H.J., Kim, J.H., Ryu, C.S., Lee, J.Y., Park, J.S., Chung, D.Y., Choi, S.Y., Kim, M.H., Chun, E.K., Roh, S.I., 2008. Protective effect of antioxidant supplementation in sperm-preparation medium against oxidative stress in human spermatozoa. Hum. Reprod. 23, 1023–1028. Fredholm, B.B., 1985. On the mechanism of action of theophylline and caffeine. Acta Med. Scand. 217, 149–153. Funahashi, H., Asano, A., Fujiwara, T., Nagai, T., Niwa, K., Fraser, L.R., 2000a. Both fertilization promoting peptide and adenosine stimulate capacitation but inhibit spontaneous acrosome loss in ejaculated boar spermatozoa in vitro. Mol. Reprod. Dev. 55, 117–124. Funahashi, H., Fujiwara, T., Nagai, T., 2000b. Modulation of the function of boar spermatozoa via adenosine and fertilization promoting peptide receptors reduce the incidence of polyspermic penetration into porcine oocytes. Biol. Reprod. 63, 1157–1163. Funahashi, H., Nagai, T., 2001. Regulation of in vitro penetration of frozen-thawed boar spermatozoa by caffeine and adenosine. Mol. Reprod. Dev. 58, 424–431. Gadella, B.M., Tsai, P.-S., Boerke, A., Brewis, I.A., 2008. Sperm head membrane reorganisation during capacitation. Int. J. Dev. Biol. 52, 473–480. Harrison, R.A.P., Gadella, B.M., 2005. Bicarbonate-induced membrane processing in sperm capacitation. Theriogenology 63, 342–351. Krueger, C., Rath, D., Johnson, L.A., 1999. Low dose insemination synchronized gilts. Theriogenology 52, 1363–1373. Kunavongkrit, A., Sang-Gasanee, K., Phumratanaprapin, C., Tantasuparuk, W., Einarsson, S., 2003. A study on the number of recovered spermatozoa in the uterine horns and oviducts of gilts, after fractionated or non-fractionated insemination. J. Vet. Med. Sci. 65, 63–67. Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A., Parrilla, I.J., Vazquez, L., Day, B.N., 2001. Successful non-surgical deep intrauterine insemination with small numbers of spermatozoa in sows. Reproduction 122, 289–296. Martinez, E.A., Vazquez, J.M., Roca, J., Lucas, X., Gil, M.A., Parrilla, I.J., Vazquez, L., Day, B.N., 2002. Minimum number of spermatozoa required for normal fertility after deep intrauterine insemination in non-sedated sows. Reproduction 123, 163–170. Matthijs, A., Engel, B., Woelders, H., 2003. Neutrophil recruitment and phagocytosis of boar spermatozoa after artificial insemination of sows, and the effects of inseminate volume, sperm dose and specific additives in the extender. Reproduction 125, 357–367. Noguchi, M., Yoshioka, K., Itoh, S., Suzuki, C., Arai, S., Wada, Y., Hasegawa, Y., Kaneko, H., 2010. Peripheral concentrations of inhibin A, ovarian steroids, and gonadotropins associated with follicular development throughout the estrous cycle of the sow. Reproduction 139, 153–161. Noguchi, M., Yoshioka, K., Hikono, H., Iwagami, G., Suzuki, C., Kikuchi, K., 2015. Centrifugation on Percoll density gradient enhances motility,
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Please cite this article in press as: Noguchi, M., et al., Effect of semen extenders on frozen-thawed boar sperm characteristics and distribution in the female genital tract after deep intrauterine insemination in sows. Anim. Reprod. Sci. (2015), http://dx.doi.org/10.1016/j.anireprosci.2015.11.006