The experiment was conducted at the Geode Gene Transfer Center boar stud (TriOak Foods, Inc., IA, USA). Boars were housed in individual stalls on slatted floors over a deep pit in an air-conditioned barn with a positive pressure air filtration system. Fresh water, sourced from a public (rural) water supply, was provided by individual nipple waterers and feed was supplied individually each morning based on the body condition score (BCS) of each boar. Using a BCS ranking system of 1 - 5, a BCS of 3+ is considered ideal, and 3.2 kg of feed per boar per day was provided to maintain boars at this level. Boars with a lower BCS were fed additional feed and boars with a higher BCS were fed slightly less.

Boars were Pig Improvement Company (PIC, Hendersonville, TN, USA) terminal sires developed for optimum growth performance and carcass quality. PIC 337 (Pietrain cross) and PIC 800 (Duroc) lines were used in the study. Three groups of 87 boars per group, representing all of the mature boars available at the time the study was run, were assigned with each group made up of 54 PIC 337 and 33 PIC 800 boars. A power test was not run to determine the number of boars required to answer these questions prior to the initiation of the trial. Boars were assigned to treatment to equalize age, average sperm production in the previous 12 weeks and BCS.

Boars received a corn-soybean meal diet that was balanced for net energy and formulated to meet or exceed requirements for amino acids, minerals and vitamins as specified by NRS [1]. All boars were maintained on the control diet before the start of the study for a minimum of 12 weeks (Table 1). The control diet provided 1.83 g of DHA per boar per day from a commercially available marine omega-3 fatty acid supplement, fed according to the manufacturer’s recommendations. The 3 test diets contained an omega-3 supplement composed of refined fish oils, vitamin E and hydrophobic starch (Salmate®, The Ballard Group Inc., Cincinnati, OH, USA) at an inclusion level calculated to provide 1.83 g, 2.38 g and 2.94 g per boar per day of DHA for test diets 1, 2 and 3 respectively.

For the first part of the experiment, test diets 1 through 3 were compared to determine if the level of inclusion of supplemental DHA via the Salmate® ingredient influenced semen volume, sperm concentration and sperm quality characteristics in the 2 boar lines. The source of the marine fatty acids in the boar stud diet was changed to Salmate® on 10-Aug., 2020 and all boars received the test diet from 10-Aug., 2020 until completion of the trial on 02-Nov., 2020. For test diets 2 and 3, boars received the additional Salmate® via top-dress at 8 g and 16 g per boar per day, respectively. Semen samples collected between 12-Oct., 2020 and 02-Nov., 2020 were included in the analysis, allowing a 9 week period of exposure to the product for sperm cellular growth and development.

^aOmega 3 sources supplied 1.83 g DHA per boar per day for each diet; ^bVitamin-mineral premix, synthetic amino acids, choline chloride, and phytase were isogenous for both diets.

For the second part of the study, production data from 77 of the 87 boars on the control diet (previous DHA source) were compared to data from the same boars on test diet 1, with both diets providing 1.83 g DHA per boar per day. Boars were eligible for inclusion if they had received the control diet for a minimum of 9 weeks prior to collection of semen parameter data, and were in production through 02-Nov., 2020. Data collected from 14-June, 2020 through 05-July, 2020 were used to represent the control feeding period. Semen collected between 12-Oct., 2020 and 02-Nov., 2020 from the same boars was evaluated as the test feeding period.

Semen was collected by the gloved hand technique from boars previously trained to mount dummy sows. Ejaculate volume was determined by weight. Within 2 minutes of completion of ejaculation, a sample of the raw semen was diluted at a 1:14 ratio with AndroStar Plus® semen extender (Minitube USA, Inc., Verona, WI, USA) and processed through an eFlow® chamber to determine sperm concentration and quality (Androvision® Computer Assisted Sperm Analysis System (CASA), Minitube USA, Inc., Verona, WI, USA). Once these measurements were completed, the data were automatically transferred into Prism® software (Minitube USA, Inc., Verona, WI, USA). The CASA analysis yielded data on sperm concentration, motility, both progressive and non-progressive, and gross morphology, which included percent proximal and distal cytoplasmic tail droplets as well as translocated tail abnormalities. Findings were automatically transferred into Prism® software and recorded. From these results, the program determined the number of semen doses to make at 2.25 × 10⁹ total sperm per dose for ejaculates that met minimum requirements for customer use.

Ejaculates were required to meet the following criteria to be accepted for use:

1) Combined percent of morphological defects (proximal and distal cytoplasmic droplets and translocated tails) could not exceed 25%.

2) Total motility must be >80% and progressive motility must be >70%.

3) Minimum doses per ejaculate must be >5.

Feed samples for each of the diets were analyzed by Eurofins, Des Moines, IA, USA to determine the level of fatty acids supplied by the diets. This analysis of the average value of DHA level in the feed samples was within 7% variance of calculated levels of DHA.

For sperm quality parameters, statistical analysis was conducted using a general linear model determined through stepwise regression and included the fatty acid treatment, the 2 genetic lines of boars, the age of the boars and the number of days rest between the collection of semen. The analysis was carried out using Minitab 16 Statistical Software® (Minitab LLC, State College, PA, USA). Chi-square analysis was conducted to assess differences between ejaculate rejection rates. A two tailed test with Yates corrected was used.

Nine of the boars originally assigned to treatment were not available for the duration of the trial. Two boars in each of treatments 1 and 2 died, and 5 boars from treatment 1 were moved out of the program by facility management. The remaining 159 PIC 337 boars averaged 824 days of age, with a range from 332 to 1341 days of age. For the PIC 800 boars, 93 boars remained that averaged 538 days of age, with a range from 366 to 1192. The number of days between semen collections averaged 6.84 ± 3.01 for the PIC 337 line and 6.01 ± 2.86 days for the PIC 800 boars. In the final analysis, there were 51 PIC 337 and 29 PIC 800 boars in test diet 1, 54 PIC 337 and 31 PIC 800 boars in test diet 2, and 54 PIC 337 and 33 PIC 800 boars in test diet 3.

Results showing the effects of the 3 dietary levels of Salmate® are provided in (Table 2). There were noteworthy differences between the 2 genetic lines for this phase of the research. Sperm concentration was greater for the PIC 800 line (P < 0.001), while ejaculate volume was greater with the PIC 337 line (P < 0.001). Other differences were not significant (P > 0.2) between the lines. There were no differences in the individual sperm quality parameters measured that could be attributed to the dietary treatments.

Mean of 2.25 × 10⁹ cells/dose.

There were no diets by genetic line interactions for any of the parameters measured.

Findings for semen rejection parameters are provided in (Table 3). The table shows the incidences of both the rejected as well as the retained occurrences for each measurement and when combined, they add up to the total number of samples within each treatment. The overall rejection rate was low with all treatments. While there were no differences in the individual semen quality parameters, the intermediate treatment level resulted in fewer ejaculates being rejected due to low motility (P < 0.026) and fewer total ejaculates being discarded (P < 0.045). This overall increase in the percent of ejaculates accepted for treatment 2 equates to 7.5% and 6.4% more ejaculates processed compared to the lowest and highest levels of DHA inclusion, respectively.

Ten of the originally assigned boars were not available at the end of the trial and were not included in either data set. Two died, 3 were not available for the entire control period and the remaining 5 boars were transferred out of the program by facility management. The remaining were 49 PIC 337 and 28 PIC 800 boars. For this part of the trial, the PIC 337 boars averaged 762 days of age, with a range of 251 to 1340 days of age. The PIC 800 boars averaged 482 days of age, with a range of 247 to 1133 days of age. The PIC 337 boars averaged 6.76 ± 1.38 days rest between collections while the PIC 800 boars averaged 6.06 ± 1.49 days rest. Results (Table 4) showed that there were no differences in the extent of sperm abnormalities between the 2 dietary fatty acid sources [proximal cytoplasmic droplets (P = 0.33), distal cytoplasmic droplets (P = 0.68), translocated tail abnormalities (P = 0.21)]. However, the concentration of sperm was lower (P < 0.023) for the boars during the test period (Salmate®) compared to the control feeding period. This was offset by a greater volume of semen per ejaculate during the test feeding period (P < 0.001), resulting in a greater number of total sperm cells (P < 0.001) and significantly more insemination doses being produced per ejaculate (P < 0.001).

Results in Part 2 of this study (Table 5) show that there were no statistical differences in the number of rejected ejaculates between the Salmate® treatment and the previous source of DHA with all P values greater than 0.17 for each of the parameters measured.

¹GLM included treatment, boar line, and number of days of rest; ²Treatment *Line interaction; ³Mean of 2.25 × 10⁹ cells/dose.

There were no effects of DHA level fed on the individual semen quality parameters measured (P > 0.15) in this trial (Table 2). However, (Table 3) clearly shows that the overall ejaculate rejection rate was significantly lower (P < 0.045) for the intermediate level of Salmate® compared to the lowest and highest levels of inclusion, resulting in 7.5% and 6.4% more ejaculates passing minimum standards for processing into semen doses. It has been found previously that the concentration of DHA was lower in low motility boar sperm [10], but these researchers accepted 60% motility as adequate, which is much lower than this trial. It has also been determined that DHA improved semen volume and total sperm numbers per ejaculate, much as occurred in Part 2 of this trial, but did not influence motility when 80% was used as the cut-off point for rejected ejaculates [11]. The fact that there was no additional response to the highest inclusion level of DHA warrants further evaluation of the 3 levels of DHA investigated in this study to determine whether higher levels of DHA coupled with adjustments to the diet could have a larger impact on semen quality parameters, ejaculate rejection rates, and/or total semen production than were observed in this trial.

When the trial was designed, it was not known if the PIC 800 (Durocs) would have a higher requirement for DHA, as suggested by previous work [8]. The lack of an interaction between genetic lines and DHA feeding levels in this trial indicates that this is not the case. One limitation of this trial is there was no negative control group that did not receive supplemental DHA in the diet. Additional research is also warranted to further evaluate potential performance differences between the Duroc line tested in this study and other Duroc lines.

These results clearly demonstrated a significant advantage (P < 0.001) for the stabilized marine lipid supplement (Salmate®) which increased total sperm output per ejaculate over the control in Part 2 of this experiment. While the motility and progressive motility remained unchanged for these boars, the volume of semen and the total number of sperm cells per ejaculate increased with the treatment regimen, providing a clear economic advantage to this collection facility. It has been previously reported that boar diets containing supplemental algae rich in DHA increased semen volume without altering semen quality parameters [11].

All results presented are for the same boars and demonstrate an improvement in total sperm output when the stabilized fatty acid supplement (Salmate®) was substituted for the DHA product previously used by this facility. It can be argued that the time periods for Part 2 of the trial were different, potentially favoring the Salmate® product. However, this should have worked against the treatment feeding program. It was previously found that sperm concentration, volume and number of potential doses of semen declined with declining photoperiod [12]. Day length was reduced for the treatment group of boars compared to the controls in this trial.

Age was rejected as a factor in the regression model due to the short time between sampling periods. In an extensive meta-analysis of over 250,000 records, it was determined that age influences semen production, but only over the long term, with optimum age being 3.5 years for maximum sperm output for boars [13]. Due to housing conditions, heat stress would not have been a factor in this study. PIC 800 boars were included in the trial because a previous publication showed that omega-3 supplemented diets positively affected both sperm morphology in Large White and Pietrain breeds as well as the osmotic resistance of Pietrain spermatozoa [8]. No effects were seen in the Duroc boars in that study. However, in this trial, the PIC 800 (Duroc) line responded positively to the treatment program with an increase in total sperm output.

As with Part 1 of this trial, there is the limitation that no negative control was included that would demonstrate differences between the two DHA products added and no DHA in the diet.

Salmate® significantly reduced the percent of rejected ejaculates when fed at 2.38 g DHA per boar per day, compared to 1.83 g and 2.94 g, by 7.5% and 6.4%, respectively, in mature boars in a commercial boar stud in two terminal genetic lines. Further investigation is warranted to determine if increasing levels of this protected DHA product (Salmate®) coupled with other dietary adjustments would result in further increases in sperm output, improved sperm quality and/or further reductions in the percent of rejected ejaculates. Salmate® also significantly increased total sperm output without altering sperm cell quality or the percent of rejected ejaculates when compared to another commercially available product when both provided 1.83 g DHA per boar per day. Although sperm concentration was reduced, total volume, and thus, the total number of sperm cells increased per ejaculate by 17%.