Seminal attributes and sperm morphology of Agonda Goan pigs

Abstract

Study of local breeds and their inclusion in breeding programme is essential owing to their higher disease resistance and adoptability to stresses. Agonda Goan, a local pig breed found in Goa, India, is preferred due to its suitability in acclimatization, less feed requirement and capacity to utilize locally available fibrous feeds. Seminal attributes namely, semen volume, sperm concentrations per millilitre and total sperms per ejaculate and the sperm morphology in Agonda Goan pigs was studied. Average semen volume was 87.31 ml and total sperms per ejaculate were 10.79 × 10⁹ which were less as compared to mean semen volume (204.00 ml) and total sperms per ejaculate (111.63 × 10⁹) in Large White Yorkshire pigs. Mean head area and mean sperm length of the Agonda Goan pig were less than that of sperms of Large White Yorkshire. These observations add to the scientific knowledge as this demonstrates differences in sperm size between boars of different breeds.

Keywords:

• Agonda Goan pig
• sperm morphology
• bacteria in semen
• seminal attributes

1. Introduction

Smallholder resource-driven pig production system is economically viable and sustainable at household level (Kumaresan et al. 2009). Local breeds and low input rearing practices such as low cost housing, feeding with available feeds and fodders play an important role in the sustainability of smallholder pig production system. In India pig rearing is developing as an enterprise in areas of North Eastern Hilly region, Kerala, Goa and Bihar. Though Goa is the smallest of India it shows an astonishing diversity of endemic species, habitats and ecosystems. In Goa amongst livestock, pigs are the most important and people rear pigs as a backyard venture due to abundant availability of hotel waste due to tourism industry. Agonda Goan is a local pig breed of the state traditionally known as ‘Gavthi Dukor’ (village pig). This breed is known for its adaptability and ability to thrive well with available feed resources. Agonda Goan is a small body statured, early maturing pig Figure 1 (Chakurkar 2009) with rough black bristles. Females are good mothers Figure 2 (Chakurkar et al. 2013). Pork of Agonda Goan pig is preferred by local sausage industry. This has attracted its conservation and use in crossbred production. Study of local breeds and their inclusion in breeding programme is essential as they have higher disease resistance and adoptability to stress. Boar prolificacy depends on the quality of generated reproductive cells which can be determined by examining sperm morphology. Males can differ with respect to the frequency of spermatozoa with morphological abnormalities (Phillips et al. 2004; Kondracki & Wysokińska 2005).

Assessment of sperm morphology is considered as a major component of the spermiogramme as it relates to fertility of the ejaculate (Saravia et al. 2007). Visual-motility assessment is subjective by nature which is used to determine the quality of boar ejaculates (Tejerina et al. 2008). Subsequently, for more objective assessment using conventional image analysis or particle counting, computer-assisted sperm analysis (CASA), with different software designs has been developed (Tejerina et al. 2008). The use of artificial insemination (AI) in the swine industry has increased exponentially and most of the semen is still used as liquid, extended and stored at temperatures slightly below room temperature (17–20°C). During the semen collection process, it is difficult to maintain a sterile procedure. Therefore, bacterial contamination is common in freshly collected boar ejaculates (Sone 1990; Althouse & Lu 2005), the majority being the Gram-negative bacteria. Besides the boar, other sources of bacterial contamination include environment, personnel and the water used for extender preparation (Althouse & Lu 2005). Bacteria may exert their spermicidal effect directly upon the sperm cell. Consequently, there is an increasing interest in the diagnostic methods used for semen analysis in AI. The objective of the present was to study the seminal attributes of Agonda Goan pigs and its comparison with Large White Yorkshire.

2. Materials and methods

2.1. Experimental animals

Experimental animals included six sexually mature Agonda Goan and four Large White Yorkshire boars maintained for breeding. The mean age of Agonda Goa pigs was of 23.33 ± 5.14 months and that of Large White Yorkshire was 25.66 ± 2.74 months. Mean body weight of Agonda Goan boars was 38.91 ± 5.71 kg, whereas mean weight of Large White Yorkshire was 195.66 ± 7.08 kg.

2.2. Management of animals

Breeding boars were housed individually in well-ventilated pens with average temperature range of 25–30°C. Animals were fed with the standard breeder mixture containing maize, rice polish, soybean meal, mineral mixture and common salt, as ingredients to fulfil the nutrient requirements (Nutrient Requirements of Swine: 10th Revised Edition 1998). Feeding was done by offering the ration twice daily in equally divided doses after moistening with water in semi-solid form, while clean drinking water was made available throughout the day.

2.3. Testicular (scrotal) dimensions

Thickness and length of both side scrotal sac and testis was measured to get idea of testicular size. Length and thickness were measured using Vernier calliper. These observations indicated difference in size (volume) of testicles (Table 1).

Table 1. Age, weight and scrotal sac dimensions of Agonda Goan and Large White Yorkshire pigs.

	Agonda Goan pigs	Large White Yorkshire pigs
Parameter	Mean ± SEM	Mean ± SEM
Age (months)	23.33 ± 5.14	25.66 ± 2.74
Weight of boar (kg)	38.91 ± 5.71	195.66 ± 7.08
Length of testis (mm)	57.29 ± 12.54	131.33 ± 7.56
Thickness of testis (mm)	36.24 ± 1.58	67.26 ± 2.99

2.4. Semen collection

Ejaculates were collected with ‘gloved-hand’ method (Hancock & Hovell 1959) over the period of four months. Due to shy breeding and wild nature of the Agonda Goa males, sow in oestrous was used as dummy for the semen collection. For Large White Yorkshire boars, dummy sow (IMV, France) was used for semen collection. Sperm rich fractions were used for semen analysis. A total 36 ejaculates from Agonda Goan and 36 ejaculates from Large White Yorkshire boars were used for the study.

2.5. Semen evaluation

Semen attributes like total volume, sperm concentration per millilitre of semen and total sperms per ejaculate were calculated. For total sperm counting Accucell photometer (IMV) was used. Abnormal sperm counts were also studied using Hamilton Thorne CASA (IMV) in which coiled tail, bent tail, distal mid-piece reflex (DMR), distal droplet and proximal protoplasmic droplets were calculated. Sperm morphology characters like head length, head width, head perimeter, head area, tail length and total length were analysed in 900 sperms from Agonda Goa and 450 sperms from Large White Yorkshire boars using CASA (Ceros II) system.

2.6. Bacterial count

Total bacterial load and micro flora in neat semen were studied. Fifteen semen samples (randomly collected) were analysed for the presence of bacterial contamination by plating them on blood agar and on brilliant green agar. Blood agar was also used for the isolation of Staphylococcus species and Streptococcus species. Brilliant green agar allows differentiation of Enterobacteriaceae including Escherichia coli, Serratia species, Enterobacter species, Proteus species and Klebsiella species. For bacterial growth, the plates were incubated at 37°C for 18–24 h.

2.7. Statistics

The data were analysed statistically for test of significance (Snedecor & Cochran 1994) by using WASP 2.0 software.

3. Results and discussion

Sperm output significantly differs across breeds and their crossbreds. The highest heterosis effect for semen volume was 30.6%, for number of total spermatozoa was 18.2% and for number of viable spermatozoa was 10.4% (Nwakpu & Onu 2007). Sperm output varied with season, including high values in autumn and winter and low ones in spring and summer (Smital et al. 2004). Mean length of testis from outside the scrotum in Agonda Goan boar was 57.29 ± 12.54 mm, whereas in case of Large White Yorkshire the mean length was 131.33 ± 7.56 mm. Thickness of the testis from outside the scrotal sac was 36.24 ± 1.58 and 67.26 ± 2.99 mm in case of Agonda Goan and Large White Yorkshire, respectively. The mean body weight of Agonda Goan boar was 38.91 ± 5.71 kg, whereas the Large White Yorkshire boars weighed 195.66 ± 7.08 kg. The breeding value of boar depends of the quantity and quality of semen produced. Ejaculate variation is the product of an influence of many factors including race (Smital et al. 2004; Smital 2009), season of the year (Wysokinska et al. 2009), boar age (De Vos et al. 2003), testicular size (Oyeyemi et al. 2002; Oyeyemi and Okediran 2007) as well as utilization intensity (Frangez et al. 2005). Mean volume of the sperm rich fraction was 87.30 ml in case of Agonda Goan, whereas this volume was 204.00 ml in case of Large White Yorkshire boar. Boar prolificacy depends on the quantity and quality of generated reproductive cells which can be determined by examining sperm morphology. The results of such an examination make it possible to check fertility of a male and its suitability for crossbreeding (Phillips et al. 2004). Sperm concentration per millilitre of semen in case of Agonda Goan boar was 136.80 × 10^6, whereas sperm concentration in case of Large White Yorkshire was 559.46 × 10⁶. In Agonda Goan, mean total sperms per ejaculate were 10.79 × 10^6, whereas mean total sperms per ejaculate in case of Large White Yorkshire were 111.63 × 10⁶ (Table 2). This difference attributes to variation in size of testis in these breeds. Although sperm concentration is low as compared to Large White Yorkshire, the values are in acceptable limit (Larsson 1986; Wiseman et al. 1998). The number of the abnormalities influences the fertilizing ability of sperm, which is synonymous with boar prolificacy (Colenbrander & Kemp 1990). Total sperm abnormalities in case of Agonda Goan boar semen were 16.38%, whereas total abnormalities in Large White Yorkshire boar semen were 10.64%. Although the difference was significant (p < 0.001) the abnormalities were within acceptable limits. Bent tail and coiled tails were 4.12% and 3.11% in Agonda Goan boar semen. In case of Large White Yorkshire, these abnormalities were 1.88% and 1.06% (Table 3). There was no significant difference in DMR and distal droplet per cent, and both were in acceptable limits. Proximal droplet per cent was slightly higher in Agonda Goan than Large White Yorkshire, i.e. 3.10% vs. 2.03%. In the present study, normal sperm count is in agreement with that reported earlier (Kondracki et al. 2012), the percentages of normal spermatozoa in case of Pietrain and Duroc reported (Kondracki et al. 2012) were 94.02 ± 5.64 and 93.53 ± 4.28, respectively. Pietrain had 1.39 ± 1.94% and 4.59 ± 4.49% of primary and secondary abnormalities, respectively, while it was 1.06 ± 2.02% and 5.41 ± 4.03% for Duroc (Kondracki et al. 2012).

Table 2. Seminal attributes of Agonda Goan and Large White Yorkshire pigs.

	Agonda Goan pigs	Large White Yorkshire pigs
Parameter	Mean ± SEM	Mean ± SEM
Volume	87.30 ± 7.57*	204.00 ± 13.21*
Concentration	136.80 ± 18.57×10^6*	559.46 ± 56.68×10^6*
Total of sperms per ejaculate	10.79 ± 1.53×10^9*	111.63 ± 14.20×10^6*

Note: n = 36.

* P < 0.01.

Table 3. Sperm abnormalities in Agonda Goan and Large White Yorkshire pigs as analysed by CASA.

	Agonda Goan pigs	Large White Yorkshire pigs
Morphological abnormalities (%)	Mean ± SEM	Mean ± SEM
Bent tail	4.12 ± 0.7*	1.88 ± 0.24*
Coiled tail	3.11 ± 0.58*	1.06 ± 0.17*
DMR	2.83 ± 0.36	2.44 ± 0.29
Distal droplet	3.21 ± 0.37	3.20 ± 0.39
Proximal droplet	3.11 ± 0.42**	2.03 ± 0.24**
Total abnormalities	16.38 ± 1.66*	10.64 ± 0.88*

Note: n = 36.

* P < 0.01; **P < 0.05.

Table 4. Sperm morphology of Agonda Goan and Large White Yorkshire pigs.

	Agonda Goan (n = 900)	Large White Yorkshire (n = 450)
Morphological parameters	Mean ± SEM	Mean ± SEM
Head length (µm)	7.19 ± 0.03*	7.501 ± 0.03
Head width (µm)	2.56 ± 0.01**	2.569 ± 0.02
Head perimeter (µm)	17.45 ± 0.08**	17.945 ± 0.08
Head area (µm)^2	15.96 ± 0.11*	16.387 ± 0.20
Tail length (µm)	19.38 ± 0.12*	21.015 ± 0.12
Total length (µm)	26.57 ± 0.14*	28.526 ± 0.14

* P < 0.01; **P < 0.05.

Figure 1. Agonda Goan Male.

Figure 2. Agonda Goan female with litter.

Dimension in addition to spermatozoa livability and motility is important factors of post copulation semen selection. Spermatozoa dimensions may be associated with sperm numbers (Wysokinska et al. 2009). Spermatozoon dimensions can depend on sperm concentration in ejaculate (Pinart et al. 1998; Rijsselaere et al. 2004). Sperm head is the genetic information carrier during fertilization process so its size and shape may play important role in this process (Kondracki et al. 2012). Sperm head is associated with chromatin structure and integrity, male prolificacy is influenced by sperm morphology (Ostermeier et al. 2001). Head dimensions like head length, head width, head perimeter and head area in Agonda Goan (Table 4) were significantly less than that of dimensions of Large White Yorkshire. Spermatozoa of highly fertile boars have got smaller and shorter heads compared to spermatozoa of boars that are less effective in terms of fertilising performance (Hirai et al. 2001). Tail length and total length of sperm of the Large White Yorkshire boar were significantly more than that of sperm of Agonda Goa. Observations of present study are comparable with that reported earlier (Kondracki et al. 2012) wherein Peitrain spermatozoa have longer tails than Duroc which indicated that they were more motile as spermatozoon velocity was dependent on length (Gomendio & Roldan 1991). Total length of Pietrain spermatozoa was 53.72 ± 1.79 µm and that of Duroc was 53.20 ± 1.35 µm. Head length and tail length of Pietrain pig sperm were 9.20 ± 0.39 and 44.52 ± 1.72 µm, respectively, and for Duroc it was 9.41 ± 0.31 and 43.72 ± 1.09 µm, respectively. Head width, perimeter and area of sperm head were 4.65 ± 0.25 µm, 23.82 ± 1.25 µm, 40.47 ± 2.72 µm^2, respectively, in case of Pietrain and 4.80 ± 0.18 µm, 24.17 ± 0.88 µm, 40.73 ± 1.18 µm², respectively, for Duroc breed (Kondracki et al. 2012). Tail length is associated with a different amount of energy produced in mitochondria which results in different motility of sperms (Bierła & Giżejewski 2007).

Microbiological results indicated the growth of a variety of bacteria from different genera. Thirty-three per cent of the samples contained two or more different bacterial genera. The most frequently isolated bacteria from porcine extended semen were from both enteric (Enterobacter, E. coli, Serratiamarcescens) and non-enteric (Alcaligenes). Staphylococcus aureus was also isolated. Earlier study (Althouse & Lu 2005) reported Enterococcus spp., Stenotrophomonasmaltophilia, Alcaligenesxylosoxidans, Serratiamarcescens, Acinetobacterlwoffi, E. coli, Pseudomonas spp. and others. It has been suggested that prudent individual hygiene, good overall sanitation and regular monitoring can contribute greatly in controlling bacterial load. Incorporation of temperature-dependent bacterial growth and hyper thermic augmentation of antimicrobial activity are valuable for effective control of susceptible bacterial loads.

4. Conclusion

In conclusion, sperm concentrations per millilitre and total sperms per ejaculate in Agonda Goan pigs were almost one third than that of Large White Yorkshire breed but are in acceptable range as per suggested limit value for boar semen. Mean head area and mean sperm length of the Agonda Goan pig were less than that of sperms of Large White Yorkshire. These observations add to the scientific knowledge as this demonstrates differences in sperm size between boars of different breeds. Further studies on the suitability of semen ejaculates of Agonda Goan pig to preserve at refrigeration temperature as well as at ultra-low temperature to be carried out which is an essential step for dissemination of quality germplasm to different parts of the state if required.

Acknowledgement

The authors are thankful to Indian Council of Agricultural Research and Director, ICAR Research Complex for Goa, Old Goa for providing the necessary facilities to carry out the research work.