
==== Front
Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

39289411
72070
10.1038/s41598-024-72070-2
Article
Temporal effect of flaxseed oil in boar’s diet on semen quality, antioxidant status and in-vivo fertility under hot humid sub-tropical condition
Singh Mahak mahaksinghivri@gmail.com

1
Mollier R. Talimoa 1
Kumar Dinesh 2
Katiyar Rahul 3
Chamuah J. K. 4
Kumar Sunil 5
Chaudhary J. K. 6
Deori Sourabh 3
Kalita H. 1
Mishra V. K. 3
1 https://ror.org/023azs158 grid.469932.3 0000 0001 2203 3565 Animal Reproduction Laboratory, ICAR Research Complex for NEH Region, Nagaland Centre, Medziphema, Nagaland 797106 India
2 Department of Animal Science, (Animal Nutrition), College of Agriculture, Jawaharlal Nehru Krishi Vishwa Vidyalaya, Tikamgarh, M.P. 472001 India
3 https://ror.org/023azs158 grid.469932.3 0000 0001 2203 3565 ICAR Research Complex for NEH Region, Umiam, Meghalaya 793103 India
4 https://ror.org/043w1y866 grid.465029.c 0000 0004 1762 1313 ICAR-National Research Centre On Mithun, Nagaland Centre, Jharnapani, Medziphema, Nagaland 797106 India
5 https://ror.org/03pczck54 grid.506011.3 ICAR-National Research Centre On Pig, Rani, 781131 Guwahati, Assam India
6 grid.459438.7 0000 0004 1800 9601 Department of Animal Genetics and Breeding, College of Veterinary Sciences & Animal Husbandry, Central Agricultural University (CAU), Selesih, Aizawl, Mizoram 796014 India
17 9 2024
17 9 2024
2024
14 216949 10 2023
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
The present study investigates the temporal effects of flaxseed supplementation on boar semen quality, antioxidant status, and in-vivo fertility under high-temperature humidity index (THI) conditions in a sub-tropical climate. Twelve Hampshire crossbreed boars were randomly assigned to control and treatment groups, with the treatment group receiving flaxseed oil supplementation. Semen samples were collected and analyzed for semen quality parameters, sperm kinematics, and antioxidant status. Fertility outcomes were assessed through in-vivo mating trials. Flaxseed supplementation resulted in time dependent significant improvements in semen volume, sperm concentration, total and progressive sperm motility, sperm quality parameters, and antioxidant status. Fertility outcomes, including farrowing rates and litter sizes, were also enhanced in the flaxseed-supplemented group. These findings highlight the potential of flaxseed supplementation to improve boar fertility under high ambient stress conditions, with implications for optimizing reproductive performance in swine production systems.

Keywords

Boar fertility
Flaxseed supplementation
Semen quality
Sperm kinematics
Antioxidant status
Sub-tropical climate
Subject terms

Reproductive disorders
Infertility
issue-copyright-statement© Springer Nature Limited 2024
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pmcIntroduction

Pigs are more susceptible to environmental heat stress because of anatomical and physiological reasons. The thermo-neutral zone of pig is 18 °C to 25 °C only. In adult pigs above 110 kg body weight, the thermo-neutral zone is further reduced below 21 °C hence, they are more susceptible to environmental heat stress1,2. With increasing global temperature, the risk of heat stress for pig is also growing. Climate induced heat stress affects behavioral, physiological, immunological and reproductive functions of pigs3. These changes become more evident in adverse climate where intensity and frequency of heat stress is more. In tropical and sub-tropical climate, high humidity and high temperature is a major constraint for pig production and reproduction3,4. Under such climate, the long duration of high temperature humidity index period severely jeopardize the pig welfare and reproduction. It has been reported earlier that high temperature humidity index negatively affects the boars’ libido, semen quality and in-vivo fertility5. Heat stress during summer season is known to decrease the reproductive efficiency of boars6. The boar’s inefficient capacity to sweat, and the extensive use of temperate pigs’ breeds in sub-tropical conditions, can negatively affect the boar’s fertility7. High heat stress to boar disrupts their testis’s thermoregulation and thereby adversely affects the spermatogenesis.

Moreover, the boar scrotum is not pendulous7,8 and boar spermatozoa tend to be more susceptible to temperature shock9. Stone10 reported that spermatogenesis in boars is impaired when ambient temperatures rise above 29 °C. Heat stress in boars has been shown to result in lower semen volume, reduced sperm concentration3,11 lower motility and higher rates of abnormal spermatozoa, interference in testosterone production, reduced libido and decreased in-vivo fertility3,4,12. It was earlier reported that heat stress induces DNA damage in boar spermatozoa during spermatogenesis which may contribute significantly to seasonal pregnancy failure and reduced litter size in sows13. In pigs, Didion et al.14 have proposed that spermatozoa with greater than 6% DNA fragmentation results into decreased farrowing rates and average number of piglets born. Furthermore, it was reported that there is reduced antioxidant capacity of the boar seminal plasma during the summer period3,5,15.

Besides, the boar sperm are more susceptible to damage by oxidative stress and lipid peroxidation16 because of their structural composition which becomes more pronounced during summer season when the ambient temperature is high17,18. Boar sperm membrane has a unique composition with low cholesterol to phospholipids ratio and therefore more susceptible to cold shock during liquid preservation. In today’s swine reproduction, artificial insemination with liquid preserved boar semen has central role in advancing the genetic gain and increasing the productivity19.

However, heat stress induced sperm damage and low keeping quality during summer months in sub-tropical climate is a significant constraint to fully reap the benefit of artificial insemination in swine reproduction20. It was reported in earlier study that boar semen quality and antioxidant status is high in autumn–winter as compared to summer season21. In tropical and sub-tropical climate, the long duration and high severity of heat stress further reduces the optimal utilization of superior genetic boar in artificial insemination programme3–5. Considering the impact of climate change and predicted heat waves, feasible strategies (managemental and nutritional supplementation) to mitigate the impact of heat stress in boars are necessary particularly in tropical climate. Recently, use of flaxseed supplementation has gained importance to mitigate the heat stress in sows and boars. Flaxseed contains 53 per cent linolenic acid which is a omega-3 fatty acid22. There are only few studied documenting the effect of flaxseed oil supplementation to boar on semen quality under tropical and sub-tropical climate3–5. Previous studies are mostly laboratory based and few have small in-vivo fertility trials. However, there is no report available on temporal effect of flaxseed oil supplementation to boar on semen quality, antioxidant status and in-vivo fertility. Therefore, the objectives of this study were (i) to determine the temporal effect of supplementing flaxseed oil on sexual behavior and semen quality parameters (SQPs) of boar sperm in humid sub-tropical climate, (ii) to evaluate effect of flaxseed oil feeding on antioxidant status and in-vivo fertility of the boar (Table 1).Table 1 Ingredients and composition of the basal diet used in the experiment.

SI No	Ingredients	Proportion (%)	CP (%)	ME (Kcal/kg)	
1	Maize	62.5	5.938	2262.5	
2	Wheat Bran	14.5	2.103	348	
3	Groundnut Cake	11	4.95	363	
4	Rice Bran	10	1.6	270	
5	Vitamin Mineral premixa	1.5	0	0	
6	Salt	0.5	0	0	
7	Total	100.00	14.59	3243.5	
a –composition (per 500 g): Ca, 23%; P, 17%; Cu, 525 mg; Co, 100 mg; I, 125 mg; Mn, 1000 mg; Fe, 1700 mg; S, 60 mg; Zn, 400 mg; Selenium, 350 mcg; Mg, 500; Niacin, 500 mg; Methionine, 2100 mg; Lysin, 2500 mg; Choline, 250 mg; vitamin A, 1,70,000 i.u.; vitamin B1, 8 mg; vitamin B2, 100 mg; vitamin B6, 10 mg; vitamin B12, 50 mg; vitamin D3, 60,000 i.u.; and vitamin K, 100 mg.

Results

Effects of flaxseed supplementation to boar on sexual behavior and semen quality parameters at day 0

There was significant effect of feeding flaxseed oil on reaction time and false mount from 2nd and 8th week post feeding, respectively (Table 2). Similarly, sperm viability, sperm acrosomal integrity and HOST reactive spermatozoa were significantly higher in flaxseed group from 8th weeks of flaxseed oil feeding. Sperm abnormality decreased significantly (P < 0.05) in flaxseed oil group at 6th week post treatment till 16th week.Table 2 Temporal effect of feeding linseed oil on some libido traits and semen quality parameters of boars at day 0 (mean ± SEM).

Parameters	Groups	-2wk	-1wk	0wk	1wk	2wk	4wk	6wk	8wk	10wk	12wk	14wk	16wk	
Reaction time (min)	Group-I	5 ± 0.00a	5 ± 0.26a	5 ± 0.00a	4.83 ± 0.4a	5 ± 0.00a	5 ± 0.00a	4.33 ± 0.49a	4 ± 0.45a	4 ± 0.45a	4 ± 0.45a	3.83 ± 0.4a	3.83 ± 0.4a	
Group-II	4.5 ± 0.34a	4 ± 0.45a	4.33 ± 0.21b	4 ± 0.45a	3.83 ± 0.17b	2.33 ± 0.21b	2 ± 0.00b	2.33 ± 0.21b	2.17 ± 0.17b	2.17 ± 0.17b	2.33 ± 0.21b	2.33 ± 0.21b	
False mount (numbers)	Group-I	2.33 ± 0.21a	2.33 ± 0.21a	2.17 ± 0.17a	2.33 ± 0.42a	2.33 ± 0.33a	2.00 ± 0.26a	2.33 ± 0.33a	2.17 ± 0.17a	2.17 ± 0.17a	2.17 ± 0.17a	2.33 ± 0.21a	2.33 ± 0.21a	
Group-II	2.83 ± 0.31a	2.50 ± 0.22a	2.33 ± 0.21a	2.33 ± 0.21a	2.17 ± 0.17a	1.83 ± 0.31a	1.83 ± 0.17a	1.67 ± 0.21b	1.50 ± 0.22b	1.67 ± 0.21b	1.50 ± 0.22b	1.00 ± 0.26b	
Viability (%)	Group-I	81.50 ± 0.5a	82.83 ± 0.48a	82.67 ± 0.71a	82.67 ± 0.71a	83.50 ± 0.50a	82.67 ± 0.80a	81.67 ± 0.33a	82.17 ± 0.31b	82.17 ± 0.75b	81.67 ± 1.45b	81 ± 0.52b	82.17 ± 0.83b	
Group-II	81.50 ± 0.81a	82.33 ± 0.67a	83.50 ± 1.34a	84 ± 0.63a	84.67 ± 0.99a	84.83 ± 0.70a	82.17 ± 1.17a	90.67 ± 1.38a	92.50 ± 1.06a	92.33 ± 0.67a	93.17 ± 0.75a	92.33 ± 0.76a	
Abnormality (%)	Group-I	17.67 ± 0.56a	17.17 ± 0.75a	16.17 ± 0.65a	15.67 ± 0.67a	15.67 ± 0.49a	16.33 ± 0.99a	16.67 ± 0.61a	16.50 ± 0.56a	16 ± 0.52a	15.50 ± 0.43a	15.83 ± 0.40a	16.67 ± 0.42a	
Group-II	16.67 ± 0.56a	15.83 ± 0.65a	16.17 ± 0.70a	16.67 ± 0.76a	16.33 ± 1.05a	15.50 ± 0.56a	14.67 ± 0.21b	12.83 ± 0.60b	11 ± 0.52b	12.17 ± 0.60b	11.33 ± 0.49b	11.17 ± 0.83b	
Acrosomal integrity (%)	Group-I	77.33 ± 0.56a	77.17 ± 0.70a	77.50 ± 0.34a	77.50 ± 0.67a	76 ± 0.82a	74.67 ± 0.56b	77 ± 0.63a	76.17 ± 0.60b	76.83 ± 0.70b	77.17 ± 0.87b	76.33 ± 0.88b	76.50 ± 1.02b	
Group-II	77 ± 0.82a	77 ± 0.68a	76.83 ± 0.60a	77 ± 0.68a	78 ± 0.58a	76.83 ± 0.65a	79.17 ± 0.91a	84.50 ± 0.34a	85.83 ± 0.48a	85.67 ± 0.67a	84.17 ± 0.54a	86.17 ± 0.54a	
Hypo-osmotic swelling test (%)	Group-I	75.17 ± 0.75a	74.67 ± 0.21a	75.67 ± 0.49a	74.83 ± 0.83a	74.67 ± 0.80a	73.17 ± 0.70b	76 ± 0.68a	74 ± 0.63b	75 ± 0.82b	74.17 ± 0.48b	74.50 ± 0.50b	74.17 ± 0.79b	
Group-II	74.67 ± 0.33a	74.67 ± 0.21a	74.50 ± 0.81a	75.17 ± 0.17a	74 ± 0.68a	76.17 ± 0.83a	75 ± 1.34a	80.33 ± 0.67a	80.67 ± 0.49a	80.50 ± 0.67a	79.33 ± 0.61a	80.83 ± 0.54a	
Values with different letters in superscripts in a column within a parameters express significant (P < 0.05) differences. n = 72 samples per group (12 ejaculates per boar).

Effects of flaxseed supplementation to boar on computer assisted semen analysis at day 0

Velocity attributes viz. VAP and STR% increased significantly (P < 0.05) in flaxseed group at 8th weeks post feeding till 16th week (Table 3). VSL, VCL, ALH and BCF increased significantly (P < 0.05) at 6th weeks of flaxseed oil feeding as compared to control group. LIN % increased significantly (P < 0.05) in group-II at 10th week post feeding. Total sperm motility (Fig. 5) and progressive sperm motility (Fig. 6) was significantly increased (P < 0.05) in treatment group at 6th week post feeding till 16th week.Table 3 Temporal effect of feeding linseed oil on boar semen quality parameters (CASA) at day 0 (mean ± SEM).

Parameters	Groups	-2wk	-1wk	0wk	1wk	2wk	4wk	6wk	8wk	10wk	12wk	14wk	16wk	
VAP (µm/sec)	Group-I	65.50 ± 0.81a	63.67 ± 1.28a	60.67 ± 1.86a	61.50 ± 2.20a	62.67 ± 1.89a	64.17 ± 1.30a	62.67 ± 1.89a	64.83 ± 0.79 b	65 ± 1.00b	62.50 ± 2.14b	64 ± 0.68b	65.50 ± 0.92b	
Group-II	61.67 ± 1.17b	63.67 ± 1.28a	61.17 ± 1.40a	60.67 ± 0.99a	62.83 ± 0.95a	64.17 ± 0.79a	66.17 ± 1.11a	79.67 ± 1.63a	78.83 ± 1.62a	78 ± 1.51a	77.83 ± 1.90a	78.83 ± 1.60a	
VSL(µm/sec)	Group-I	52.17 ± 1.33a	49.33 ± 2.39a	49.83 ± 1.62a	54 ± 2.59a	53 ± 1.86a	55.50 ± 1.73a	53.67 ± 1.61b	51.17 ± 2.21b	53.83 ± 0.87b	52 ± 2.10b	51.17 ± 1.87b	55.83 ± 1.99b	
Group-II	52.17 ± 2.10a	49.33 ± 1.82a	54 ± 1.51a	53.67 ± 1.84a	54.67 ± 2.11a	56.17 ± 0.83a	60.33 ± 1.89a	71.83 ± 0.91a	72.50 ± 0.89a	72.67 ± 0.80a	72.67 ± 0.84a	70.83 ± 1.54a	
VCL (µm/sec)	Group-I	123.83 ± 1.89a	126 ± 1.26a	127.83 ± 3.62a	128.83 ± 3.42a	12.33 ± 2.01a	125.17 ± 1.47a	124 ± 3.27b	124 ± 1.88b	126.83 ± 3.75b	128 ± 4.07b	120.67 ± 1.98b	129.67 ± 2.72b	
Group-II	128.33 ± 2.95a	125 ± 2.31a	124.17 ± 3.42a	123.67 ± 2.16a	118.67 ± 2.75a	123.17 ± 4.24a	139.33 ± 4.18a	158 ± 2.83a	154 ± 2.07a	152.33 ± 2.76a	152 ± 2.5a	153.67 ± 2.51a	
ALH (µm)	Group-I	5.67 ± 0.49a	5.33 ± 0.56a	5.50 ± 0.34a	5.50 ± 0.50a	5.33 ± 0.42a	6.17 ± 0.75a	4.83 ± 0.40b	5.50 ± 0.56b	5.33 ± 0.33b	5.17 ± 0.60b	5.67 ± 0.21b	5.83 ± 0.40b	
Group-II	5.33 ± 0.42a	4.83 ± 0.17a	5.17 ± 0.48a	5.17 ± 0.40a	5.50 ± 0.67a	4.83 ± 0.48a	6.50 ± 0.56a	8.33 ± 0.33a	8.17 ± 0.31a	8 ± 0.37a	8.17 ± 0.31a	8 ± 0.37a	
BCF (Hz)	Group-I	30 ± 1.71a	31.17 ± 0.91a	31.50 ± 1.06a	31.33 ± 1.4a	31.50 ± 1.20a	30.67 ± 1.74a	28.33 ± 1.43b	28.33 ± 1.52b	29.67 ± 1.12b	30.17 ± 1.08b	30.83 ± 1.49b	32.17 ± 0.40b	
Group-II	30.67 ± 0.88a	30.83 ± 1.35a	29.83 ± 0.65a	29.83 ± 1.08a	30.17 ± 1.83a	27.50 ± 0.92a	41.50 ± 0.50a	40.50 ± 0.50a	42.17 ± 1.14a	42 ± 1.53a	41 ± 0.52a	40.83 ± 1.35a	
STR (%)	Group-I	79.68 ± 2.01a	77.62 ± 3.90a	82.34 ± 2.69a	87.75 ± 2.36a	84.79 ± 3.05a	86.66 ± 3.04a	86.07 ± 3.82a	79.02 ± 3.69b	82.98 ± 2.34b	83.59 ± 3.87b	79.94 ± 2.70b	85.39 ± 3.53a	
Group-II	84.88 ± 4.23a	77.61 ± 3.04a	88.51 ± 3.08a	88.50 ± 2.91a	87.24 ± 4.19a	87.60 ± 1.68a	91.34 ± 3.31a	90.33 ± 1.97a	92.15 ± 2.10a	93.33 ± 2.01a	93.56 ± 1.89a	89.99 ± 2.40a	
LIN (%)	Group-I	42.24 ± 1.60a	39.16 ± 1.92a	39.15 ± 1.68a	43.61 ± 2.97a	43.43 ± 1.90a	44.35 ± 1.31a	43.44 ± 1.80a	41.38 ± 2.17a	42.63 ± 1.43b	40.70 ± 1.47b	42.52 ± 1.97b	43.25 ± 2.14a	
Group-II	40.85 ± 2.29a	39.47 ± 1.25a	43.75 ± 2.17a	43.45 ± 1.58a	46.26 ± 2.33a	45.86 ± 1.61a	43.36 ± 1.08a	45.56 ± 1.22a	47.14 ± 1.09a	47.80 ± 1.17a	47.90 ± 1.24a	46.13 ± 1.07a	
Values with different letters in superscripts in the same row express differences which were significant (P < 0.05). VAP: Average Path Velocity, VSL: Straight line Velocity, VCL: Curve linear Velocity, ALH: Amplitude of lateral head displacement, BCF: Beat cross frequency, STR: Straightness, LIN: Linearity. n = 72 samples per group (12 ejaculates per boar).

Effects of flaxseed supplementation to boar on semen volume and sperm concentration

Semen volume increased significantly (P < 0.05) in group-II (flaxseed) from 2nd week post feeding till 16th week as compared to the control group (Fig. 3). Sperm concentration increased significantly (P < 0.05) from 6th week onward in flaxseed group as compared to control group (Fig. 4). In overall, flaxseed oil supplementation resulted in significant enhancement of boar semen production and improvement in semen quality parameters during summer months.

Effects of flaxseed supplementation to boar on semen quality parameters in liquid stored boar semen after 72 h of storage

After liquid storage of semen for 72 h, sperm livability and acrosomal integrity were significantly (P < 0.01) higher in flaxseed group (group-II) as compared to control group (group-I) from 6th weeks onward (Table 4). Abnormal spermatozoa were significantly lesser (P < 0.01) in flaxseed group as compared to control group from 6th weeks onward. HOST positive sperm were significantly (P < 0.01) increased in flaxseed group as compared to control group from 8th weeks onward (Table 4).Table 4 Temporal effect of feeding linseed oil on boar semen quality parameters after 72 h of storage (mean ± SEM).

Parameters	Groups	-2wk	-1wk	0wk	1wk	2wk	4wk	6wk	8wk	10wk	12wk	14wk	16wk	
Viability (%)	Group-I	76.33 ± 0.61a	75 ± 0.45a	74.33 ± 0.21a	74.67 ± 0.95a	73.33 ± 0.61a	73 ± 0.77a	74.33 ± 0.49b	73.83 ± 0.60b	73.83 ± 0.1.08b	74.50 ± 0.22b	73.50 ± 0.50b	73.67 ± 0.56b	
Group-II	73.67 ± 1.12a	74.67 ± 0.92a	73.33 ± 1.09a	73.17 ± 0.70a	72.17 ± 0.91a	72.83 ± 0.70a	77.67 ± 0.67a	81.67 ± 0.21a	82 ± 0.63a	83.17 ± 0.75a	81.83 ± 1.38a	81.17 ± 0.65a	
Abnormality (%)	Group-I	19.33 ± 0.71a	19.33 ± 1.45a	19.33 ± 1.23a	19.33 ± 0.88b	19.83 ± 0.54a	20.33 ± 1.09a	20.83 ± 0.83a	22.17 ± 0.54a	20.83 ± 0.70a	20 ± 0.45b	20.33 ± 0.61a	20 ± 0.73a	
Group-II	20.83 ± 0.48a	19.83 ± 0.65a	21 ± 0.52a	22.50 ± 0.62a	20.33 ± 0.71a	20.50 ± 0.56a	15.83 ± 0.87b	16 ± 0.58b	15.50 ± 0.56b	14.50 ± 0.34a	15.33 ± 0.33b	16 ± 0.63b	
Acrosomal integrity (%)	Group-I	70.67 ± 0.67a	71.67 ± 0.67a	70.50 ± 0.81a	72 ± 1.57a	71.17 ± 1.08a	70 ± 1.24a	71.67 ± 0.92a	70.50 ± 1.15b	71.83 ± 0.70b	71.83 ± 0.54b	71 ± 0.37b	71.50 ± 0.43b	
Group-II	70.50 ± 0.62a	72.17 ± 0.40a	71.83 ± 0.48a	72.50 ± 0.34a	73.17 ± 0.70a	72.67 ± 0.42a	71.67 ± 0.56a	80.50 ± 0.43a	80.83 ± 0.60a	81 ± 0.63a	80.17 ± 0.75a	79.67 ± 0.76a	
Hypo-osmotic swelling test (%)	Group-I	64.83 ± 0.98a	63.17 ± 0.60a	63.33 ± 0.61a	63.50 ± 0.56b	63.33 ± 0.67a	64 ± 0.89a	63.50 ± 0.67a	63.50 ± 0.43b	64.33 ± 0.92b	62.67 ± 0.49b	64 ± 1.00b	63.67 ± 0.88b	
Group-II	65 ± 0.26a	63.83 ± 0.83a	64.83 ± 0.79a	65.50 ± 0.56a	65.33 ± 0.61a	64.33 ± 0.61a	65 ± 0.93a	73 ± 0.63a	74.33 ± 0.61a	73.67 ± 0.61a	73.33 ± 0.92a	74.17 ± 0.48a	
Values with different letters in superscripts in the same row express differences which were significant (P < 0.05). n = 72 samples per group (12 ejaculates per boar).

Effects of flaxseed supplementation to boar on computer assisted semen analysis of liquid stored boar semen after 72 h of storage

After 72 h of storage, total sperm motility and progressive sperm motility were significantly increased (P < 0.05) in treatment group at 6th week post feeding till 16th week (Table 5). Similarly velocity attributes viz. VAP, VSL, VCL, ALH and BCF were significantly (P < 0.01) higher in flaxseed group as compared to control group at 6th week onward of flaxseed oil feeding.Table 5 Temporal effect of feeding linseed oil on boar semen quality parameters (CASA) after 72 h of storage (mean ± SEM).

Parameters	Groups	-2wk	-1wk	0wk	1wk	2wk	4wk	6wk	8wk	10wk	12wk	14wk	16wk	
Total motility (%)	Group-I	63.83 ± 0.48b	64.67 ± 0.67a	64.50 ± 0.76a	64.17 ± 0.79a	64.17 ± 0.60a	64.50 ± 0.67a	65.50 ± 0.67b	64.67 ± 0.56b	65.83 ± 0.60b	64.50 ± 0.92b	62.67 ± 0.67b	64.17 ± 0.54b	
Group-II	66.17 ± 0.48a	63.67 ± 0.49a	64.67 ± 0.84a	65.17 ± 0.48a	65 ± 1.03a	64.17 ± 0.70a	72 ± 1.06a	74.50 ± 0.22a	75.17 ± 0.31a	74.33 ± 0.56a	74.33 ± 0.21a	76 ± 0.77a	
Progressive motility (%)	Group-I	33.83 ± 0.65a	34 ± 0.68a	35.17 ± 0.79a	34.17 ± 0.70a	35 ± 0.63a	33.83 ± 0.98a	34 ± 0.63b	34.50 ± 1.20b	34.17 ± 0.60b	33.33 ± 0.71b	33.83 ± 1.19b	35 ± 1.03b	
Group-II	36 ± 1.13a	34.67 ± 0.42a	34 ± 1.13a	32.50 ± 0.56a	33.33 ± 0.61a	33.67 ± 0.76a	40 ± 0.58a	42.17 ± 0.54a	43 ± 0.52a	44 ± 1.00a	44.83 ± 0.83a	43.33 ± 0.76a	
VAP (µm/sec)	Group-I	55.6 ± 0.76a	57 ± 0.68a	55.83 ± 0.75a	56 ± 0.89a	55 ± 0.68a	54.83 ± 0.91a	57 ± 0.82b	53.83 ± 0.95b	58.33 ± 0.92b	56.50 ± 0.72b	55.17 ± 0.83b	56 ± 1.00b	
Group-II	55.17 ± 0.79a	54 ± 0.86b	54.83 ± 0.70a	53.67 ± 0.92a	54 ± 0.68a	55 ± 1.21a	62.67 ± 0.71a	67 ± 0.77a	70.67 ± 1.78a	68.67 ± 1.15a	67.67 ± 1.05a	69.83 ± 1.83a	
VSL(µm/sec)	Group-I	43.67 ± 1.65a	43.17 ± 2.15a	43.17 ± 1.45a	42.67 ± 1.26a	45.67 ± 2.42a	44.83 ± 2.47a	43.33 ± 2.36b	43.33 ± 2.08b	42.33 ± 0.95b	42.17 ± 1.68b	41.67 ± 2.49b	43 ± 1.90b	
Group-II	43.33 ± 0.92a	44 ± 1.03a	43.83 ± 1.01a	43.50 ± 1.06a	43.17 ± 1.08a	42.83 ± 1.08a	55.33 ± 0.61a	58.50 ± 1.41a	57.83 ± 2.63a	59.50 ± 1.48a	58 ± 0.93a	58 ± 1.21a	
VCL (µm/sec)	Group-I	105 ± 3.57a	103.67 ± 5.78a	106 ± 4.74a	102.83 ± 3.99a	97.50 ± 4.03a	98.83 ± 2.15a	96 ± 1.57b	97.50 ± 2.40b	101.67 ± 3.11b	102.67 ± 2.55b	101.67 ± 3.04b	104.50 ± 4.26b	
Group-II	101.67 ± 2.84a	104.17 ± 3.76a	107.33 ± 3.32a	105.83 ± 4.00a	103 ± 3.85a	97.83 ± 2.50a	138.50 ± 1.31a	140.33 ± 1.43a	137.83 ± 3.25a	140.67 ± 2.32a	145.67 ± 3.90a	137.67 ± 3.43a	
ALH (µm)	Group-I	4.17 ± 0.40a	4.17 ± 0.17a	4.17 ± 0.40a	3.50 ± 0.43a	4.83 ± 0.17a	4.33 ± 0.33a	3.83 ± 0.40b	3.83 ± 0.60b	4.17 ± 0.31b	3.50 ± 0.43b	4.17 ± 0.48b	3.83 ± 0.40b	
Group-II	4.50 ± 0.56a	4.67 ± 0.33a	4.17 ± 0.31a	4.50 ± 0.22a	5 ± 0.58a	4.50 ± 0.22a	6 ± 0.37a	6 ± 0.52a	6.0 ± 0.52a	5.00 ± 0.26a	6 ± 0.52a	6 ± 0.37a	
BCF (Hz)	Group-I	25 ± 0.45a	24.50 ± 0.43a	24.67 ± 0.76a	24.83 ± 0.17a	25.50 ± 0.89a	24.50 ± 0.34a	24.83 ± 0.48b	24.33 ± 0.42b	25.50 ± 0.92b	25.17 ± 0.65b	25.83 ± 0.60b	25.67 ± 0.80b	
Group-II	25.33 ± 0.71a	24.50 ± 0.22a	24.17 ± 0.31a	24 ± 0.26b	25 ± 0.52a	24.50 ± 0.22a	33.17 ± 0.79a	33.83 ± 0.60a	34 ± 0.82a	32 ± 0.73a	32.17 ± 1.62a	32.67 ± 1.31a	
STR (%)	Group-I	78.67 ± 3.84a	75.91 ± 4.27a	77.43 ± 3.05a	76.35 ± 2.88a	80.03 ± 3.75a	82.12 ± 5.43a	76.17 ± 4.66b	80.90 ± 4.95a	72.69 ± 2.17a	74.59 ± 2.58b	75.40 ± 3.95b	76.83 ± 3.26a	
Group-II	78.59 ± 1.66a	81.56 ± 2.08a	80.05 ± 2.41a	81.06 ± 1.38a	79.95 ± 1.80a	78.24 ± 3.50a	88.38 ± 1.68a	87.41 ± 2.60a	82.18 ± 4.53a	86.73 ± 2.24a	85.75 ± 1.15a	83.36 ± 2.90a	
LIN (%)	Group-I	42.05 ± 2.93a	42.37 ± 3.37a	41.11 ± 2.12a	41.80 ± 1.93a	47.67 ± 1.46a	45.64 ± 3.16a	45.15 ± 2.34a	44.49 ± 2.11a	41.83 ± 1.56a	41.09 ± 1.35a	41.12 ± 2.60a	41.37 ± 1.99a	
Group-II	42.71 ± 0.98a	42.45 ± 1.53a	40.93 ± 0.92a	41.32 ± 1.50a	42.28 ± 2.20a	43.89 ± 1.39a	39.95 ± 0.30a	41.72 ± 1.19a	42.08 ± 2.12a	42.33 ± 1.08a	40 ± 1.48a	42.34 ± 1.84a	
Values with different letters in superscripts in the same row express significant differences (P < 0.05). VAP: Average Path Velocity, VSL: Straight line Velocity, VCL: Curve linear Velocity, ALH: Amplitude of lateral head displacement, BCF: Beat cross frequency, STR: Straightness, LIN: Linearity. n = 72 samples per group (12 ejaculates per boar).

Effects of flaxseed supplementation to boar on antioxidant status in seminal plasma and spermatozoa

Seminal plasma GPx and TAC were significantly (P < 0.01) elevated following flaxseed oil supplementation as compared to control group from first week onwards (Table 6). Seminal plasma MDA decreased significantly (P < 0.01) in flaxseed group as compared to control group from first week post feeding till 16th weeks. However, spermatozoa MDA level decreased significantly in flaxseed group from 6th weeks onward only.Table 6 Temporal effect of feeding linseed oil on boar semen antioxidant parameters and lipid peroxidation at fresh stage (mean ± SEM).

Parameters	Groups	-2wk	-1wk	0wk	1wk	2wk	4wk	6wk	8wk	10wk	12wk	14wk	16wk	
Seminal plasma GPx (nmol/min/mL)	Group-I	76.83 ± 1.35a	79 ± 1.71a	78.17 ± 2.10a	75.67 ± 0.76b	77 ± 1.61b	76.50 ± 1.73b	77.83 ± 1.99b	76.17 ± 1.58b	75.17 ± 1.72b	80.67 ± 2.20b	76.67 ± 1.28b	76.50 ± 2.32b	
Group-II	77.50 ± 1.23a	78.67 ± 1.78a	81.83 ± 0.75a	88.67 ± 1.76a	108 ± 2.08a	113 ± 0.82a	110 ± 1.39a	112 ± 2.80a	107 ± 1.26a	111.50 ± 1.93a	112.50 ± 1.23a	109.67 ± 2.64a	
Seminal plasma TAC (mmol/L)	Group-I	0.28 ± 0.01a	0.29 ± 0.01a	0.28 ± 0.01b	0.28 ± 0.01b	0.29 ± 0.01b	0.31 ± 0.01b	0.30 ± 0.01b	0.29 ± 0.01b	0.30 ± 0.01b	0.30 ± 0.01b	0.29 ± 0.01b	0.28 ± 0.01b	
Group-II	0.29 ± 0.01a	0.30 ± 0.01a	0.32 ± 0.01a	0.32 ± 0.01a	0.38 ± 0.00a	0.38 ± 0.01a	0.38 ± 0.01a	0.36 ± 0.01a	0.38 ± 0.01a	0.38 ± 0.01a	0.37 ± 0.01a	0.38 ± 0.01a	
Seminal plasma MDA (nmol/mL)	Group-I	1.67 ± 0.02a	1.64 ± 0.03a	1.68 ± 0.02a	1.70 ± 0.04a	1.67 ± 0.03a	1.63 ± 0.03a	1.71 ± 0.02a	1.68 ± 0.02a	1.64 ± 0.03a	1.69 ± 0.03b	1.66 ± 0.02a	1.74 ± 0.02a	
Group-II	1.72 ± 0.01a	1.67 ± 0.02a	1.63 ± 0.02a	1.55 ± 0.01b	1.43 ± 0.02b	1.34 ± 0.02b	1.37 ± 0.03b	1.36 ± 0.03b	1.33 ± 0.03b	1.36 ± 0.05a	1.33 ± 0.04b	1.37 ± 0.02b	
Spermatozoa MDA (nmol/108 sperm cells)	Group-I	3.62 ± 0.08a	3.73 ± 0.05a	3.72 ± 0.06a	3.68 ± 0.05a	3.70 ± 0.05a	3.66 ± 0.07a	3.68 ± 0.06a	3.58 ± 0.05a	3.70 ± 0.09a	3.66 ± 0.04a	3.67 ± 0.07a	3.69 ± 0.06a	
Group-II	3.66 ± 0.03a	3.66 ± 0.03a	3.63 ± 0.02a	3.66 ± 0.02a	3.64 ± 0.01a	3.65 ± 0.06a	2.94 ± 0.11b	2.89 ± 0.02b	3.08 ± 0.03b	3 ± 0.05a	3.06 ± 0.04b	2.95 ± 0.05b	
Values with different letters in superscripts in the same row express significant differences (P < 0.05). GPx: Glutathione peroxidase, TAC: Total antioxidant capacity, MDA: Malondialdehyde. n = 72 samples per group (12 ejaculates per boar).

Effects of flaxseed supplementation to boar on in-vivo fertility

Significantly (P < 0.05) higher farrowing rate was recorded with semen of boars supplemented with flaxseed oil as compared to control group (Table 7). Litter size at birth and litter size at weaning was significantly (P < 0.05) higher in flaxseed group (group-II) as compared to control group (group-I).Table 7 Effect of feeding linseed oil to boar on in-vivo fertility (mean ± SEM).

	Sows inseminated(n)	Animal farrowed (n)	Farrowing rate (%)	Litter size at birth (mean ± SEM)	Litter size at weaning (mean ± SEM)	Pre-weaning mortality (%)	
Group-I	198	140	70.70b	9.85 ± 0.14b	8.44 ± 0.10b	14.34	
Group-II	140	115	82.14a	10.96 ± 0.18a	9.71 ± 0.14a	12.21	
Values with different letters in superscripts in the same column express significant differences (P < 0.05). n = 198 pluriparous sows in the control group and 140 pluriparous sows in treatment group.

Discussion

It has previously been reported that boar semen is affected by season, temperature and photoperiod which in turns impact reproductive performance4,7,31–33. During the course of the present experiment, boars were under very high environmental stress as evident by high temperature humidity index which caused poor semen quality in control group. It was reported that high temperature-humidity index compromises sperm quality and fertility of Holstein bulls34 and boars5,11,35. A greater incidence of sperm morphological abnormalities, reduced sperm motility and compromised fertility of boars was reported at higher ambient temperatures36.

The present experiment, recorded for the first time the temporal effect of flaxseed oil supplementation to the boar diets on semen quality and reproductive performance in sub-tropical climate under high environmental stress condition. The results of the present study indicate that flaxseed supplementation had temporal effects (positive) on the semen quality parameters and sperm kinematics at fresh stage as well as after 72 h of liquid storage. Besides, feeding flaxseed oil further improves the antioxidant status of the boar as well as enhances the in-vivo fertility.

In the present study, significant effect of feeding flaxseed oil on reaction time and false mount from 2nd and 8th week post feeding, respectively was recorded. Similarly, sperm quality parameters and CASA attributes were significantly improved from 8th weeks of flaxseed oil feeding at the fresh stage as well as after 72 h of storage at 17 °C. However, sperm abnormality decreased significantly in flaxseed oil group at 6th week post treatment. In the present study, significantly higher semen volume was obtained from second week onward where as increased sperm concentration was recorded from sixth week onward of the flaxseed oil supplementation. Increased boar semen volume and also of sperm concentration by flaxseed supplementation during high environmental stress period will produce more functional sperm per ejaculates. This will in turn increase the reproductive efficiency of genetic superior boars by inseminating more sows with a single collection3. According to the results of the present study, the flaxseed oil supplementation to boar has to be started at least six weeks prior to summer season to optimize the production of good quality fertile sperm. Liu et al.25 reported positive effect of PUFAs supplementation to boars at six weeks after treatment. In the literature, there is an inconsistent report regarding effects of PUFA supplementation to boar on its semen quality and fertility. However, the present study recorded a clear temporal response of feeding flaxseed oil and improvement in semen quality parameters in boars during high ambient stress. M.J.Estinne et al.37 reported increased sperm concentration of boar after dietary supplementation with omega-3 fatty acid there was no improvement in spem morphology. Similarly, Maldjian et al.38 reported that PUFAs through tuna fish oil supplementation to boar diets increased sperm output. In another study, it was observed that dietary ratio of n-6:n-3 is important to have positive outcome and 6.6:1 ratio was found to improve progressive sperm motility in boars25. Castellano et al.39 reported improvement in liquid stored semen quality of boars after tuna fish oil supplementation but no effect was observed on cryopreserved semen. It was also reported that supplementation of flax meal to the boars' diet improved the quality of fresh as well as cryopreserved boar semen40. In another study, supplementing bull diets with flaxseed oil significantly increased total motility, progressive motility and motion characteristics of cryopreserved sperm, however, no effect was observed at the fresh stage41. In stallion, dietary supplementation of linseed oil plus antioxidants improved cooled–stored stallion semen quality, however, it did not improve cryopreserved semen42. The difference with the other studies might be because of differences in climatic condition, intensity of environmental stress, breed (genetic selection for heat tolerant AI-boars), duration of feeding and source of omega-3 fatty acid4,11,43. Supplementation with dietary linseed oil during peri-puberty is known to stimulate steroidogenesis and testis development in rams44.

The improvement in semen quality parameters and fertility after the flaxseed oil supplementation may be because of the lowering of lipid peroxidation in spermatozoa40 as observed in present study also. Yan et al.45, observed a positive effect of supplementation with n-3/n-6 PUFAs with ratio of 1:1 in boars on testicular development and spermatogenesis. Polyunsaturated fatty acids affect the hypothalamic-pituitary–gonadal axis which in turn improves the endocrine profiles, testicular development and spermatogenesis. In rat, higher n-3 fatty acid in diet increased the concentrations of GnRH, FSH, LH and testosterone45 which in turn increased fertile sperm production. It was also postulated that n-3 fatty acids integrates into the spermatozoa membrane and alter the sperm membrane lipid profile and thereby improves sperm quality and fertility46. In agreement with our findings, Perumal et al.47 observed significant improvement in mithun bull’s semen under the similar climatic condition of high THI after flaxseed oil supplementation. It was suggested that dietary flaxseed oil (high in n-3 PUFA) supplementation possibly improved cholesterol to phospholipid ratio of sperm plasma membranes, altered the affinity and expression of gonadotropin receptors and upregulated the testosterone synthesis mechanism47. It can be suggested that PUFAs present in flaxseed oil are involved in the flagellar movement of sperm which in turn increases sperm motility and fertility47. Similar results were reported in other species like buffalo48,49 stallion42 ram44 and cattle50. In addition, feeding flaxseed oil might have improved the semen quality by increasing the antioxidant status of the boars’ semen and spermatozoa under the stressful condition of high THI.

The antioxidant defense of the boar semen and sperm in control group was in compromised state during high THI period. In case of treatment group, flaxseed oil supplementation to boar significantly alleviate oxidative stress during high ambient temperature humidity index period thereby improved the semen quality and fertility. Seminal plasma GPx, TAC and MDA were improved following flaxseed oil supplementation from first week onwards. However, spermatozoa MDA level decreased significantly in flaxseed group from 6th weeks onward only. The finding suggests that antioxidant milieu of seminal plasma improved at the earliest followed by that of spermatozoa post flaxseed oil supplementation. Mammalian sperm are prone to oxidative stress owing to their limited antioxidant capacity and this in turn adversely affects sperm survival, impair their functions such as motility, membrane integrity and fertilizing ability51. Among farm animals, boar sperm are more prone to oxidative stress, because of the high proportion of PUFAs in the membrane and lower cholesterol to phospholipid ratio, which can be easily oxidized leading to lipid peroxidation52. Oxidative damage in sperm occurs due to increase production of reactive oxygen substances (ROS) and/or a decreased natural antioxidant defense system more particularly in adverse climatic condition like high ambient temperature53. Spermatozoa have limited antioxidant defenses to protect itself from oxidative damage of ROS. Heat stress may induces DNA strand breaks, fragmentation of spermatozoa DNA and spermatozoa chromatin packaging defects, germ cell apoptosis in testis, incomplete epididymal spermatozoa maturation, and increased exposure to ROS54. The negative impact of heat stress on spermatozoa DNA integrity is coupled with its downstream effect on early embryo development13. However, seminal plasma protects the spermatozoa from oxidative damage and keeps ROS-levels within the physiological range, compatible with the functional life of the sperm55. In agreement with our findings, Perumal et al.47 reported reduced oxidative damage in mithun spermatozoa after flaxseed oil supplementation in subtropical climatic conditions. Dietary n-6:n-3 fatty acid in 6.6 ratio significantly improved antioxidant status in boar serum, sperm and seminal plasma25. Strzezek et al.3,5,56 reported similar results in boar seminal plasma. Diet enriched with transgenic flax improves the quality of fresh and conserved boar semen by lowering the lipid peroxidation in boar spermatozoa40. Flaxseed oil may have protected spermatozoa because of its antioxidant and/or free radical quenching properties. This property of flaxseed oil is because of its constituent bioactive ingredients such as omega-3 fatty acids and lignans57. Lignans has antioxidant property in quenching the free radical and it inhibits peroxyl-radical-mediated damage of DNA58. It was reported that the presence of polyphenols and vitamin E may also have contributed to its antioxidant property or it stimulates the activities of antioxidant enzymes47. However, further study is needed to better understand flaxseed oil role in improving the antioxidant defense mechanism of semen and spermatozoa.

Despite all of the above discussed finding, till now there is no single boar semen quality test which can be used as a prospective indicator of their in-vivo fertility59. Therefore, in-vivo fertility study was done in this experiment to validate the hypothesis that flaxseed oil improves boar sperm fertility. In-vivo fertility results revealed higher fertility of semen of boars supplemented with flaxseed oil under high THI. Farrowing rate improved significantly (increased by 17%) with semen of boars supplemented with flaxseed oil as compared to control group. This was an interesting finding as it recorded a greater quantitative difference between flaxseed supplemented and control group. Also, the more numbers of sows inseminated conclusively validate the positive effect of flaxseed oil supplementation on improved boar fertility during high environmental stress period. Similarly, litter size at birth and litter size at weaning was significantly higher in flaxseed group. The pre-weaning mortality was statistically similar in both groups. In our previous study, we reported improvement in in-vivo fertility but not in litter size at birth after flaxseed oil supplementation, however, that study included very small numbers of sows3,4. Increased in-vivo fertility following flaxseed oil supplementation could be due to improved sperm quality parameters, antioxidant status and fertilizing ability both at the fresh stage as well as after 72 h of storage. With increased in antioxidant capacity, spermatozoa from boars supplemented with flaxseed oil would be more adapted to survive in the oviductal environment until the moment of fertilisation. The number of total embryos that a sow can produce after an ovulation does not depend on the total number of ovulated oocytes, but rather on the number of oocytes that are optimally fecundated31. Therefore, sperm of flaxseed supplemented boars would have a greater fertilizing ability, thus increasing the number of fully functional embryos yielded. The positive correlation between sperm quality and field fertility has been reported previously60,61. Singh et al.3,4 reported significantly higher in-vivo fertility of sows breeding by AI using semen from flaxseed oil-fed boar. However, in another study, in-vivo fertility of boars did not improve after cod liver oil supplementation as a source of PUFAs62. Excessive reactive oxygen species production in boar semen more particularly during environmental stress compromise sperm membrane functions, effuses cholesterol from sperm membrane, damage DNA and mitochondrial membrane potential, thereby, resulting in poor fertility outcome31,63. Supplementation of flaxseed oil might have inhibited all these processes in spermatozoa and thereby improved in-vivo fertility. Although the findings of the study have implications for improving the pig production system in sub-tropical climate, however, it applicability in other climatic condition needs to be evaluated further. Besides, long term study are needed to assess the sustainability of the observed improvements and to evaluate any potential adverse effects associated with prolonged flaxseed supplementation.

In conclusion, the present study has demonstrated for the first time that dietary supplementation of flaxseed oil to boar improved the semen quality parameters and sperm kinematics in time dependent manner under high environmental stress condition. It also improved total antioxidant capacity and minimized lipid peroxidation in seminal plasma as well as in sperm. Besides, flaxseed supplementation significantly improved in in-vivo fertility (in terms of farrowing rate and litter size) of semen of boar fed flaxseed oil. Therefore, it can be concluded that flaxseed oil may be incorporated in boar’s diet before six weeks of adverse climate or season for improvement of semen quality and fertility in a sub-tropical climate. Nevertheless, further large studies in other breeds are warranted for delineation of the exact mechanism by which flaxseed oil supplementation improves sperm quality and fertility.

Methods

Location of study and climatic parameters

The study was approved by Institute Animal Ethics Committee of ICAR Research Complex for North Eastern Hill Region, Umiam, Meghalaya, India (2100/GO/RBi/L/20/CPCSEA dated 19.05.2020) and all methods were performed in accordance with the relevant national guidelines and regulations. The study was conducted in compliance with ARRIVE guidelines. No anaesthetic agent was used in the present study. After completion of experiment, animals were continously used in the farm for routine breeding programme. The experiment was conducted at Pig Research Farm of ICAR Research Complex for NEH Region, Nagaland Centre, Medziphema, Nagaland, India from 13th May to 22nd September. The research farm is located at latitude of 25°45' N, the longitude of 93°50' E and altitude of 281 m above mean sea level. The climate of the region is hot-humid sub-tropical with annual rainfall varies from 1500 to 2000 mm. Temperature and humidity data were collected by an automatic weather station of Gramin Krishi Mausam Seva of India Metrological Department (IMD) located at ICAR Research farm close to Pig Research Farm. The following equation was used to calculate the THI3,23 where T is the temperature in degrees Celsius and RH is relative humidity.THI=0.8×T∘C+RH%/100×T(∘C)-14.4+46.4

The weakly mean THI was calculated as the mean of the daily mean THI values. During the study period, the minimum and maximum temperature humidity index was 77.21 and 91.99, respectively (Fig. 1). The THI indicates that boars were under severe heat stress as reported earlier by Singh et al.3,5.Fig. 1 Maximum and minimum temperature humidity index (THI) during the study period (mean ± SEM).

Animals

Twelve Hampshire crossbreed (50% Hampshire and 50% Gunghroo) boars aged 18 to 24 months were included in this study. The average body weights of the animals were 150.16 ± 3.43 and 149.66 ± 3.67 kg in control and treatment group, respectively. Boars were housed in individual concrete pens (9 m2 area) with the provision of the open area (4 m2 area); however, no cooling facilities were present; therefore, temperatures were similar inside and outside of the pen. Experimental animals were maintained under uniform feeding, lighting, housing and other standard managemental practices as per the farm schedule.

Experimental design

Boars were randomly divided into control (Group-I) and treatment (Group-II) with six boars in each group (Fig. 2). Animals were fed a corn and groundnut cake diet (Table 1) and the boars were restricted to 3.0 kg feed/day (3). The diet composition was based on the nutritional requirements according to NRC24. The boars were provided ad libitum access to drinking water. In control and treatment group, vegetable oil (canola) and flaxseed oil was top-dressed at the rate of 3.0 per cent (90 mL) in basal diets for each animal on daily basis3,4. Flaxseed or vegetable oil was given in morning and evening feeding (45 ml each time) for 16 weeks. Semen samples were collected from three weeks prior to start of treatment till 16th weeks of feeding. Semen ejaculates were collected weekly (from three weeks prior to start of treatment to two weeks post treatment) and bi-weekly from second weeks post treatment (Figs. 3, 4).Fig. 2 Experimental design of feeding of flaxseed oil to boar and semen quality, antioxidant and in-vivo fertility evaluation.

Fig. 3 Temporal effect of feeding linseed oil on boar semen volume (mean ± SEM). n = 72 samples per group (12 ejaculates per boar). Control (group-I) and Treatment (group-II) were fed vegetable oil and flaxseed oil for 16 weeks, respectively.

Fig. 4 Temporal effect of feeding linseed oil on sperm concentration of boar (mean ± SEM). n = 72 samples per group (12 ejaculates per boar). Control (group-I) and Treatment (group-II) were fed vegetable oil and flaxseed oil for 16 weeks, respectively.

Semen collection and processing for evaluating the effect of flaxseed supplementation

Semen samples were collected by the gloved-hand technique. In total, 144 ejaculates (72 ejaculates in control and 72 ejaculates in treatment group) were collected for the study (12 ejaculates per boar). Ejaculates were collected into a pre-warmed (37 °C) thermous flask. Only sperm rich fractions of the ejaculates were collected. Ejaculates were transported to laboratory for further examination and processing within five minutes of collection. After examination at fresh stage, ejaculates were diluted in isothermal Primxcell (IMV, France) media. Dilution was done so that each 80 ml semen pouch (GTB Bag manual, IMV, France) contains 3 billion motile spermatozoa. Thereafter semen pouches were stored at 17 °C in BOD incubator for 72 h. After 72 h of storage, semen pouches were warmed at 35 °C for examination. To prevent human bias in the study, the individual who collected the boar semen was not informed of feeding practices and he was not involved in laboratory semen analysis (Fig. 5).Fig. 5 Temporal effect of feeding linseed oil on total sperm motility of boar at day 0 (mean ± SEM). n = 72 samples per group (12 ejaculates per boar). Control (group-I) and Treatment (group-II) were fed vegetable oil and flaxseed oil for 16 weeks, respectively.

Evaluation of sexual behavior of boars

Reaction time was recorded in min as the time from entering collection room to the first attempt to mount the artificial sow by boar. False mounts were counted as mounting artificial sow but dismounting before semen collection by boar (Fig. 6).Fig. 6 Temporal effect of feeding linseed oil on progressive sperm motility of boar at day 0 (mean ± SEM). n = 72 samples per group (12 ejaculates per boar). Control (group-I) and Treatment (group-II) were fed vegetable oil and flaxseed oil for 16 weeks, respectively.

Evaluation of flaxseed supplementation on semen quality parameters

Semen volume was measured using a graduated cylinder. Sperm concentration was determined using haemocytometer methods25. The semen sample was diluted with 1% PBS-buffered formalin. The diluted semen was placed on a hemacytometer with the sperm counted in five squares of one chamber. Sperm heads in five squares (each square contained sixteen smaller squares) were counted in each chamber, and the counts on both sides were averaged. Semen analyses for SQPs (liveability, abnormality, acrosomal integrity, and hypo-osmotic swelling test (HOST)) were done at fresh stage and after 72 h of storage at 17 °C. The percent live spermatozoa were determined by adopting differential staining technique using Eosin-Nigrosin stain26. Spermatozoa were examined for the following abnormal morphologies: abnormal heads, abnormal tails, abnormal midpieces, detached heads, coiled tails, presence of cytoplasmic droplets27. The acrosome integrity of spermatozoa was assessed using Giemsa stain28. At least 200 spermatozoa were counted and acrosomes were considered to be intact if the entire acrosomal cap was present as viewed under oil immersion microscopy at 1000 X magnification. Spermatozoa plasma membrane integrity was evaluated by hypo-osmotic swelling test (HOST) as per29. Semen sample (100 µl) was incubated with 900 µl hypo-osmotic solution (7.35 g sodium citrate, 13.5 g fructose, in 1 L distilled water) at 37 °C for 45 min. After 45 min of incubation, eosin was added to the mixture and the mixture was spread on a warm glass slide. Dried slides were examined using light microscopy under 400 X magnification for the curled and swollen tails of 200 sperm cells in five nonconsecutive microscopic fields. Bent tails indicated that sperm cells had an intact plasma membrane.

Computer assisted semen analysis

Computer assisted semen analysis was done at fresh stage and after 72 h of storage at 17 °C as per the method described by Perumal et al.30 and Singh et al.4. Sperm kinematic parameters viz. total motility, progressive motility, average path velocity (VAP), straight line velocity (VSL), curve linear velocity (VCL), amplitude of lateral head displacement (ALH), beat cross frequency (BCF), straightness (STR) and linearity (LIN), were evaluated by Hamilton Thorne Sperm Analyser (HTM-IVOS, version IVOS 11, Hamilton Thorne Research, USA). Semen was diluted in Primxcell extender and 4 µL of this extended semen sample was placed into a pre-warmed (37 °C) chamber of disposable Leja slide (IMV, France) and was permitted to settle on the heating plate (38 °C) just prior to analysis. Five microscopic fields were analyzed for each ejaculate, in duplicates and results were presented based on the analysis of 250–400 total cells per sample.

Antioxidant analysis in seminal plasma and spermatozoa

To see the effect of flaxseed oil feeding on the antioxidant status of animals, antioxidants were estimated in seminal plasma (GPx, TAC and MDA) and spermatozoa (MDA). After collection, the semen sample of each boar was immediately centrifuged at 1000 X g/min for 15 min. Seminal plasma was stored in 2.0 mL Eppendorf (EP) tubes at -20 °C for further analysis. The sperm pellets obtained after centrifugation were re-suspended in 0.85% NaCl and washed twice by centrifugation. Aliquots of spermatozoa samples containing 3 × 108 sperm were used for the analyses of malondialdehyde (MDA). Absorbances of samples were measured by Thermo Scientific Multiskan GO Microplate Spectrophotometer, USA.

Glutathione Peroxidase (GPx) assay

Glutathione peroxidase was estimated by Cayman's Glutathione Peroxidase assay kit (703,102, Cayman Chemical Co., USA) as per the manufacturer's guidelines. This assay measures GPx activity indirectly by a coupled reaction with glutathione reductase. Oxidized glutathione, produced upon reduction of hydroperoxide by GPx, is recycled to its reduced state by glutathione reductase and NADPH. The oxidation of NADPH to NADP + is accompanied by a decrease in absorbance at 340 nm. The rate of decrease in the absorbance at 340 nm is directly proportional to the GPx activity in the sample. The absorbance was measured at 340 nm to estimate the GPx in the sample. GPx activity was expressed in nmol/min/mL. The intra and inter-assay coefficient of variation were 5.7% (n = 77) and 7.2% (n = 77), respectively.

Total antioxidant capacity (TAC) assay

Total antioxidant capacity (TAC) level (mM) was estimated using Caymen’s Antioxidant Assay Kit (Catalogue: 709,001, Cayman Chemical Co., USA) following the manufacture’s protocol. A Trolox standard curve was used to quantitate the antioxidant capacity of the sample, measured in millimolar Trolox equivalents. Absorbance was read at 750 nm. TAC concentration was expressed in mmol/L. The intra and inter-assay coefficient of variation were 3.4% (n = 84) and 3% (n = 20), respectively.

Malondialdehyde (MDA) assay

The concentration of malondialdehyde (MDA) was determined using Caymen’s thiobarbituric acid reactive substances (TBARS) assay kit (Catalogue: 10,009,055, Cayman Chemical Co., USA). Briefly, 100 µL of reconstituted MDA standard and 100 µL of samples were added in the respective five mL vial. SDS solution (100 µL) was added to each vial and swirled to mix. After that, four mL colour reagent was added forcefully and then vials were placed in boiling water for one hour. After boiling, vials were immediately placed in an ice bath for 10 min to stop the reaction. The vials were centrifuged for 10 min at 1600 X g at 4 °C and 150 µL supernatant (in duplicate) from each vial was transferred to the colorimetric plate. The absorbance was read at 540 nm. The seminal plasma MDA concentration was expressed in nmol/mL.The intra and inter-assay coefficient of variation were 5.5% (n = 10) and 5.9% (n = 8), respectively.

Malondialdehyde (MDA) assay in spermatozoa

The concentration of malondialdehyde (MDA) was determined using Caymen’s thiobarbituric acid reactive substances (TBARS) assay kit (Catalogue: 10,009,055, Cayman Chemical Co., USA) as briefed in 2.7.3. The spermatozoa MDA content was expressed as nmol/108 sperm cells.

In-vivo fertility of semen

In-vivo fertility of semen was evaluated by artificially inseminating pluriparous sow (198 with the semen of control group and 140 with the semen of treatment group on detection of oestrus as per the standard practice. To prevent human bias in the fertility outcome, the individual who did the AI in sows was not informed of feeding protocols. For in-vivo fertility, semen of control and treatment group was used from 6th weeks after start of treatment. All AIs were done within 72 h of semen collection. Farrowing rate was recorded as numbers of sows farrowed as compared to numbers of sows inseminated. Litter size at birth was recorded as actual numbers of live piglets farrowed per pig. Litter size at weaning was recorded as the number of piglets weaned at 42 days post farrowing per pig. Pre-weaning mortality was calculated as numbers of piglets weaned at 42 days compared to numbers of live piglets born. Piglets were given iron injection on 4th and 14th day of life as per standard management practices at farm.

Statistical analyses

Statistical analysis was performed using IBM Statistical Package for the Social Sciences (SPSS) v26 (SPSS Inc.; Chicago, Illinois, USA). The dataset was checked for normality and homogeneity of variances throughy Shapiro–Wilk and Levene tests, respectively. The data were analyzed using independent student t test. Farrowing rate was compared between the two groups by Chi-Square test. Square root transformation was applied to litter size and then compared by ANOVA. Results are presented as mean ± SEM with each ejaculate was considered as an independent observation, and differences were considered significant at P < 0.05.

Acknowledgements

The authors are thankful for the financial assistance received under the project “Effect of feeding linseed oil on fertility of pig and poultry under sub-tropical condition of Nagaland and Umiam” (IXX14140) of ICAR Research Complex for NEH Region, Umiam, Meghalaya, India and ICAR-All India Coordinated Research Project-Pig, New Delhi.

Author contributions

M.S. conceptualized and designed the experiment, conducted the laboratory assessments, analysed the data and contributed to write the manuscript. R.T.M. conducted the laboratory and field assessment, performed AI trials. D.K. designed the nutritional aspect of experiment and contributed to write the manuscript. R.K., J.K.C, S.K., J.K.C and S.D. analysed the data and contributed to write the manuscript. H.K. and V.K.M. designed the experiment and contributed to write the manuscript. All authors gave their final approval of the manuscript.

Data availability

All data generated or analysed during this study are included in this published article. For more information, queries may be directed to corresponding author.

Competing interests

The authors declare no competing interests.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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