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Seminal plasma inclusion during boar semen freezing improves post-thaw sperm quality in the tropics

M F Yoval-Montemira, Centurión Castro, R Ake-Villanueva, J G Magaña-Monforte, M Bottini-Luzardo1 and J C Segura-Correa1

Departamento de Reproducción Animal y Mejoramiento Genético, Facultad de Medicina Veterinaria y Zootecnia, Universidad Autónoma de Yucatán, Mérida, Yucatán, México
1 Departamento de Reproducción, Facultad de Medicina Veterinaria y Zootecnia No. 2, Universidad Autónoma de Guerrero, Guajinicuilapa, Guerrero, México


The aim was to evaluate the effect of the addition of different doses of seminal plasma (SP) during freezing on the viability of frozen-thawed boar spermatozoa. Semen from 6 boars of a commercial line was collected. The sperm-rich fraction of 30 ejaculates (5 of each boar) was obtained. Semen volume, progressive motility (PM), sperm concentration, sperm morphology, and intact acrosomes (IA) were evaluated. The treatments were: control LEY (Lactose Egg Yolk) 100% (T1), 95% LEY + 5% SP (T2), 90% LEY + 10% SP (T3), 85% LEY + 15% SP (T4), and 80% LEY + 20% SP (T5). After 2 hours of cooling at 5 °C, the semen samples were suspended in LEYGO diluent (92.5% LEY, 6% of Glycerol and 1.5% Orvus ES Paste buffer) to obtain a final concentration of 1 x 109 sperm/mL. The semen was packaged in 0.5 mL straws and frozen. The frozen semen was thawed and evaluated at 37 ºC, for PM, IA, plasma membrane integrity (PMI), and mitochondrial activity (MA).

Data were analysed using a one-way analysis of variance. The 10% SP level improved PM (52.5±2.35%) in comparison to the control (47.5±2.35%), 5% (48.0±2.35%), 15% (45.0±2.35%), and 20% SP (45.0 ± 2.35%) treatments. In addition, T3 had the highest (p<0.05) IA value (46.6±2.8%) vs T1 (36.6 ± 2.8%), T2 (40.0 2.8%), T4 (38.5±2.8%) and T5 (40.0 ± 2.8%). However, SP addition has no effect on PMI and MA.

In conclusion, the addition of 10% SP during freezing improved PM and IA but did not affect PMI and MA.

Keywords: boar, seminal plasma, frozen-thawed spermatozoa, sperm viability, semen


Frozen semen for use in artificial insemination offers various advantages such as preserving the sperm viability of animals with high genetic value for long periods, allows the establishment of semen banks, and the possibility of marketing semen doses around the world. However, porcine sperms are susceptible to frozen- thaw processes due to temperature drop, intracellular ice formation, osmotic stress and oxidative stress, affecting post-thaw sperm survival and motility (Rodríguez-Martínez et al 2008; Vilagran et al 2015; Yeste et al 2017). Therefore, sperms must be protected before and during cryoconservation.

A practice carried out in the AI industry during semen processing is the partial withdraw or the dilution of the seminal plasma (SP) and replacing it with diluents for the handling and storage of semen samples (Rodríguez-Martínez and Barth 2007; Caballero et al 2012). The reason for this is that some authors consider some fractions of the SP, such as prostatic (England and Alen 1992) and the gel fraction (Mann and Lutwak-Mann 1981), as detrimental to in vitro sperm survival. In the same way, biotechnogies such as semen freezing includes large dilutions of the sperm and subsequently partial or total withdraw of the SP, which decreases motility, metabolic activity and fertilizing capacity of the spermatozoa (Dott et al 1979; Ashworth et al 1994; Maxwell and Johnson 1999). The detrimental effect of spermatozoa function known as the dilution effect (Caballero et al 2012) has been related with dilution of the SP factors that protect the sperm from damage of the membrane and to changes similar to premature capacitation-like (Maxwell et al 1997; Maxwell et al 1998).

Various freezing protocols have been designed and different substances have been added to the freezing medium to protect spermatozoa during freezing and thawing processes (Pezo et al 2019), such as SP. SP is a composite biological fluid made of diverse natural components mixed before ejaculation; therefore, the research of its effect on spermatozoa is controversial. Some researchers have found that the exposition of spermatozoa to SP reduces the fertilizing capacity of spermatozoa (Iwamoto et al 1992), while others have shown that the addition of SP to the spermatozoa improve their fertility (Curry and Atherton 1990; García et al 2010; Heise et al 2010; Heise et al 2011). This due to, in part, to the proteins in the epididymis (Rodrigues-Martínez et al 2008); mainly due to spermadhesins PSP-I and PSP-II, probably responsible of the high rate of sperm survival during freezing (García et al 2006; Hernández et al 2007b). These specific proteins adhere to the surface of the plasma membrane providing greater stability when sperms are manipulated (Centurión et al 2003; Caballero et al 2012; Alkmin et al 2014). Recent studies (Vilagran et al 2015) suggest that the beneficial effects of SP depend on whether it comes from good freezer boar or not, even on the fraction of the ejaculate that is used. Adding 10% SP can prevent premature capacitation-like that occurs during freezing (Vadnais et al 2005). Some authors indicate that the addition of 25% SP or more to the freezing medium damages post-freeze sperm quality, while at doses lower than 25% improve the sperm quality (García et al 2010). Therefore, more studies are need about the effect of SP and the mechanisms by which SP have a protector or detrimental effect on the spermatozoa, during the process of cryopreservation.

The aim of the present study was to determine the effect of the addition of different doses of seminal plasma during the semen freezing process on the semen characteristics upon thawing.

Materials and methods

Study area

The study was carried out at the laboratory of the Department of Reproduction and Animal Breeding of the “Facultad de Medicina Veterinaria y Zootecnia de la Universidad Autónoma de Yucatán” located in Merida, Yucatan, Mexico. The climate of the region is tropical subhumid type Aw0, with annual pluvial precipitation of 983.8 mm, annual temperature of 25.8 °C (range 7-42 °C) and relative humidity between 75–80%.

Animals and semen collection and evaluation

Semen from six boars from the PIC Boar 337 commercial line was collected. The boars had 1.5 to 2 years of age, with weights of 180 to 220 kg. They were disease free and subject to the same feeding and management regimen of the farm. The boars were in an artificial insemination centre located in the central zone of the state of Yucatan, Mexico. Thirty ejaculates (5 for each boar) and obtained by the gloved hand method were used. The boars were ejaculated once a week and only the rich-fraction of the ejaculate was collected, removing the gel using filter paper. Immediately after collection, the semen was evaluated based on its macro and microscopic characteristics. The colour and appearance of the ejaculates were assessed by direct observation of the semen, and the total volume was determined by placing the semen directly into a graduated glass flask warmed at 37 °C. Progressive motility was checked in a drop of semen placed on a slide and observed at a 400 X magnification. The progressive motility was expressed as percentage according to Hernández et al (2007). Spermatozoa concentrations were evaluated putting a sample of diluted semen, in a physiological saline solution (1:100) of methylene blue and 1% formaldehyde, and with the help of a Pasteur pipette, a drop of semen was taken and placed in the Burker chamber. Spermatozoa were counted under a microscope at 400 X, and the result was expressed as the number of sperms per mL of semen.

The morphology of the sperm was determined by placing a drop of semen in a solution of 1% formaldehyde, using the technique by Pursel and Johnson (1975). A volume of 0.5 mL of the mix was deposited in a slide and the sperm morphology set on in a phase contrast microscope at 1000 X. For each sample, 200 sperms were counted, and the results expressed as percentage of normal spermatozoa.

The intact acrosomes were assessed in a solution of 1% formaldehyde (v/v), taken a drop of this solution and put down it on a slide. Smears were fixed and checked with a microscope at 1000 X, counting 200 spermatozoa in the same smear (Pursel and Johnson 1974). Spermatozoa with intact acrosomes were those that presented a dark sharp form and perfectly defined. The results were expressed as percentage.

All samples had a progressive motility  80%, sperm concentration 500 x 106 sperm/mL, sperm abnormalities <10% and 90% of intact acrosomes (Caballero et al 2012). The semen was promptly diluted in a 1:1 ratio with a BTS solution (Beltsville Thawing Solution) and placed in a refrigerator at 37 °C for transportation to the laboratory and further processing (Hernández et al 2007).

Seminal plasma

To avoid any bias due to variation in SP, it was obtained from three complete ejaculates from a different boar of the PIC Boar 337 genetic line. The boar had 2 years of age and formerly classified as a good freezer; based on sperm quality post-thawing (Watson et al 1995). Ejaculates were centrifuged trice at 3,800 g, at 17 °C per 15 min (Thurston et al 1999), and the supernatant was withdraw from the pellet and deposited in Falcón tubes. The supernatant was assessed under the microscope to warrant the total absence of sperm cells. The SP was stored at –20 °C in 1 mL aliquots until its use (Hernández et al 2007).

Freezing semen

Sperm-rich fraction of each ejaculate (n= 30) was diluted 1:1 v/v in BTS, dividing it into five equal parts. Each part was deposited in a Falcón tube (5 tubes in total) and was centrifuged at 800 g, at 17 °C for 15 min. Subsequently, the supernatant was removed and the pellet from each tube was resuspended in lactose-egg yolk diluent (LEY) or LEY + SP. The treatments were: Control, only LEY cooling diluent (100%) was added (T1); 95% LEY + 5% SP (T2); 90% LEY + 10% SP (T3); 85% LEY + 15% SP (T4); and 80% LEY + 20% SP (T5). Diluent was added to each tube with semen until reaching a concentration of 1.5 x 1099 sperm/mL. After cooling the semen during 2 hours in a refrigerator at 5 °C, the sperm were resuspended with LEYGO diluent (92.5% LEY, 6% Glycerol and 1.5 % Orvus ES Paste buffer) until a concentration of 1 x 109 sperms/mL was reached.

The semen samples were then full packed using an automatic semen packing machine (IMV, France), with each straw containing a concentration of 500 x 106 spermatozoa. The straws were deposited on a stainless grill of steel, inside a container with liquid nitrogen, so they were suspended at 5 cm from the nitrogen. Twenty minutes after the exposition to the nitrogen vapour, the straws were submerged and stored in identified baskets, in thermos with liquid nitrogen at -196 °C, where the straws were kept until assessment (Thurston et al 2001).

Thawing and semen evaluation

Four straws per treatment were thawed and quickly immersed in a water bath at 37 °C, gently shaking them for 20 seconds. The content of the four straws was poured into a test tube containing 0.5 mL of BTS (37 °C) and once the sample was mixed, the evaluation of the semen traits was carried out.

Progressive motility and intact acrosomes were evaluated as mentioned in the pre-freezing process. The fluorescence technique using carboxyfluorescein diacetate (DCF) and Rhodamine 123 (Sigma-Aldrich, St. Louis, MO, USA) determined plasmatic membrane integrity and mitochondrial activity. In this evaluation, 500 µL of each treatment was placed in a sterile vial, and transported to a dark room where 20 µL of DCF and 6 µL of Rhodamine was included. The vial was covered with aluminium foil, and incubated per 10 min in an oven, at 37 ºC. After that period of time, the sample was transferred to the dark room again and 4 µL were taken, put on a slide and covered with a coverslip to be observed in an epifluorescence microscope (Olympus, Japan) at 1,000X. Two-hundred spermatozoa were counted to determine the plasma membrane integrity, considering as cells with integral membranes, those that showed a green coloration in the head. Subsequently, the same volume (4 µL) was taken from the same sample and mitochondrial activity was evaluated, considering those that showed a green fluorescence emission in the intermediate part as sperm with mitochondrial activity (Nagy et al 2003). Results for both characteristics were given as percentages.

Progressive motility, intact acrosomes, plasma membrane integrity, and mitochondrial activity data were transformed using the square root function prior to statistical analysis. Linear, quadratic, and cubic responses of seminal plasma levels were assessed using the contrast option of the GLM procedure. In addition, means were compared using the LSMEANS option. The statistical model included treatment as a fixed and boar as a random effect. All statistical were carried out using SAS software (SAS 2012).


In this study, boar has a significant effect on all traits, except on the proportion of intact acrosomes. Table 1 shows the means and the significance of the effects of the doses of seminal plasma, added during the freezing process, on the post-thaw sperm characteristics. The PM post-thaw was higher for the treatment with 10% SP in comparison to the other treatments (p<0.05), which had similar effect (p>0.05). Similarly, the proportion of intact acrosomes was significantly greater when 10% SP was included to the semen in comparison to the other treatments. In the case of the plasma membrane integrity and mitochondrial activity, no significances were observed here among treatments (p>0.05). In addition, a quadratic effect of treatment on sperm quality was observed.

Table 1. Means + standard errors by seminal plasma (SP) doses during the freezing process on post-thaw sperm characteristics of pigs under tropical conditions


motility (%)

acrosomes (%)

Plasma membrane
integrity (%)

activity (%)

T1 (0% SP)

47.5 ± 1.84a

36.6 ± 2.76a

55.4 ± 2.05a

47.0 ± 2.96a

T2 (5% SP)

48.0 ± 1.84a

40.0 ± 2.76a

57.6 ± 2.05a

46.8 ± 2.96a

T3 (10% SP)

52.5 ± 1.84b

46.6 ± 2.76b

60.8 ± 2.05a

47.5 ± 2,96a

T4 (15% SP)

45.0 ± 1.84a

38.5 ± 2.76a

57.1 ± 2.05a

45.5 ± 2.96a

T5 (20% SP)

45.0 ± 1.84a

40.0 ± 2.76a

57.9 ± 2.05a

45.0 ± 2.96a

a,b Different letter in the same column, indicate statistical mean differences (p<0.05)


Boar differences for all characteristics were found in the present study, except for intact acrosomes. Boar is an important source of variation for semen characteristics of fresh or post-thaw semen. Roca et al (2006) concluded that inter and intra-boar variation is important on sperm concentration and morphology on fresh semen, and they, in addition, report that boar affect motility and sperm quality characteristics (plasma membrane integrity, acrosome integrity, potential of the mitochondrial membrane) in post-thaw semen. Medrano et al (2002) and Saravia et al (2005) also found boar differences on sperm quality characteristics of post-thaw semen.

The present study showed an improvement in post-thaw sperm motility with the 10% SP doses, compared to the other levels of SP and the control group. This agree with Cremades et al (2003), who detected the effect of 0 and 10% SP to the freezing diluent on post-thaw sperm survival. They note that adding 10% SP to the freezing diluent affect (p<0.05) the post-thaw sperm survival, compared to the control treatment. Those authors also observed differences (p<0.05) between SP of boar donors on sperm motility (56.7% and 59.4%) in good freezer males, compared to samples without SP (50.3%). Hernández et al (2007b) noted significant improvements in sperm motility when SP was added to the freezing medium.

Gómez-Fernández et al (2012) found an increase in sperm motility in the samples with 10 and 25% SP compared to 50% added to the freezing diluent. However, in other studies, positive effects on sperm motility after defrosting have been obtained by adding 50% SP compared to 5 and 10% but to the thawing diluent (García et al 2010; Fernández-Gago et al 2013). It was assumed that when 10% SP is added to the freezing diluent, certain components prevent the damage caused by freezing and increase sperm motility. Seminal plasma carries a family of proteins, spermadhesins, which do not bind to heparin (PSP-I and PSP-II), but bind to the surface of the sperm and protect it from processes such as high dilution or during the freezing process (Rodríguez-Martínez 2011) maintaining viability, motility and mitochondrial activity (García et al 2006). It has also been pointed out that more than 10% SP in the diluent, increases the number of factors that affect sperm viability such as the sperm motility-inhibiting factor (Kordan et al 1999), which binds to receptors of the middle piece, causing a decrease in ATP and a likely decreased in motility upon thawing.

With respect to the intact acrosomes, the percentage of them was greater in the treatment with 10% SP compared to the other treatments (p< 0.05). It is probable that, in the present study, the concentration of some components of the SP, such as some antioxidants, were present in sufficient quantity at the 10% SP doses, which allowed preventing lipid peroxidation and improving the proportion of intact acrosomes. Hernández et al (2007b) found that the addition of SP during the freezing process had a significant effect on intact acrosomes. The beneficial effects of antioxidants present in SP have been reported (Leahy et al 2010), being in part responsible for the prevention of premature freeze-induced capacitation and subsequently preventing the acrosome reaction (Vadnais and Roberts 2007). Gómez-Fernández et al (2012) observed more than 50% intact acrosomes by adding up to 25% SP to the freezing diluent. These differences between studies are probably due to variations in the proportion of SP components between semen of boars (Roca et al 2006) or between fractions of ejaculates of the same boar (Caballero et al 2004; Rodríguez-Martínez et al 2008; Saravia et al 2009).

As mentioned, in boar semen, spermadhesins are glycoproteins that represent 75% of the total proteins contained in the SP. There is evidence that the different fractions of the SP exhibit different protein profiles and therefore, some of these fractions should not be added in the preservation of semen (García et al 2006). On the other hand, there is also evidence that a certain proportion of some of these proteins can preserve the quality of frozen semen by maintaining the individual motility, sperm viability (Caballero et al 2004) and the intact acrosome membrane (García et al 2007).

Regarding the plasma membrane integrity post-thaw, no significant differences were found between doses of SP (p>0.05) as reported by Saravia et al (2009). An increase in sperms with intact membrane is the result of a greater capacity of the membrane itself for the adsorption of SP proteins, some of which may repair the damage caused by cold shock by restoring the permeability of the plasma membrane (Barrios et al 2000). Other studies mention that there is no improvement but rather detrimental effects on sperm characteristics when SP was added to the freezing medium (Moore et al 2005; Muiño-Blanco et al 2008).

In the present study, the different proportions of SP in the diluent had not effect on the mitochondrial activity (p>0.05). This agree with Pech-Sansores et al (2011) results, who evaluated the addition of SP and E vitamin to the post-freezing medium, on the mitochondrial activity in pigs. In fresh semen, the assessment of mitochondrial activity is a fair way to quantify the damage to the middle piece of the sperm, since this organelle is the place where the oxidative metabolism of the cell occurs. However, few studies have measured the effects of freezing on mitochondrial activity( Pech-Sansores et al 2011). Caballero et al (2004) mention that the incubation of boar semen, added SP from other boar could improve, decrease or inclusive have no effect on mitochondrial activity. These contentious results could be due to the seminal plasma makeup, which widely vary among boars (Muiño-Blanco et al 2008) or with the season of the year (Domínguez et al 2008).

The lack of significant differences in membrane integrity and mitochondrial activity due to the inclusion of SP to the freezing medium was probably, due to some components, such as SP epididymal proteins that strongly adhere to the plasma membrane of the spermatozoa and perhaps they could not be completely eliminated by the centrifugation of the SP (Hall et al 1996). Those proteins cannot be released or removed from the spermatic membrane, even if washed with solutions of high ionic strength. In pigs, a 27 to 28 kDa epididymal protein bind to the sperm membrane and remains until fertilization (Dacheux et al 1992). Either those proteins differ according to their properties or they can change the characteristics of the sperm membrane by smoothing the exchange of proteins, or lipids from the exterior or by modifying the composition of proteins there present (Dacheux et al 2005). It could be speculated that regardless of the proportion of SP added to the medium, certain proteins remain in the sperm membrane, even after centrifugation.

The results of the literature on the use of SP as freezing medium are contradictory. It has been observed that in boar semen classified as bad freezer, the sperm viability was higher when the SP was separated before cooling and freezing than when the sperm cells were cooled with SP and frozen without it. In contrast, the use of the good freezer boar semen may not cause significant differences between treatments (Okazaki et al 2009).



The “Consejo Nacional de Ciencia y Tecnología” for the scholarship provided to the first author.


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