Salmate DHA+ fish oil supplement fed to boars and impact on sperm production

Abstract

Omega-3 fatty acid supplementation can increase sperm production in boars, but the mechanism has not been demonstrated. The current study was to determine if the increase in sperm production was due to a reduction in germ cell apoptosis during spermatogenesis. Duroc boars (438–686 d of age) were fed either a control diet of corn, soybean meal, 5% fiber, plus vitamins and minerals per NRC requirements or the control diet plus a proprietary blend of fish oil, Salmate DHA+ supplemented diet with 1.83 g DHA (Docosahexaenoic Acid, C22:6) and 0.78 g EPA (Eicosatetraenoic acid, c20:5) omega-3 fatty acids from 26 g Salmate DHA+ (The Ballard Group, Inc. and Feedworks USA, Ltd.) per day per head added to the control diet. The control diet was fed to all boars (n = 10) for 62 d, and then Salmate DHA+ was added to the diet of 5 of the boars while the other 5 continued the control diet for another 62 d. At the end of the experiment, all boars were harvested, the testes collected, fixed with Karnovsky’s fixative, postfixed with 70% ethanol, embedded with acrylic resin, 2-micron sections taken, and sections stained with Toluidine Blue. During the study, boars were on routine semen collection. Semen data were evaluated for all boars on the first 62 d of the control diet (before period) and then for control and Salmate DHA+ boars from days 22 to 62 after Salmate DHA+ feeding started (after period). A total of 107 ejaculates were evaluated in the study. The control boars’ total sperm per ejaculate (×10⁹) was 105.7 ± 7.3 vs. 110.9 ± 10.4 in the before vs. after period (p > 0.05). The Salmate DHA+ boars’ total sperm per ejaculate (×10⁹) was 103.4 ± 5 vs. 122.8 ± 7 in the before vs. after period (1.18-fold increase, P < 0.05). To examine apoptosis via histology, the number of round spermatids (RS) and Sertoli cells (SC) were counted in 10 stage 1 seminiferous tubules for each boar with adjustments made for cell nucleoli or nuclei size and thickness of the histology section. The ratio of RS/SC or Sertoli Cell Index was then determined to estimate how many RS survived apoptosis in spermatogenesis. The Sertoli Cell index was 9.46 ± 0.84 vs. 15.03 ± 0.97, respectively, for Control and Salmate DHA+ boars (P < 0.01). The number of RS/SC increased 1.59-fold due to Salmate DHA+ treatment and demonstrates a reduction in apoptosis. The increased number of RS may explain the increased sperm per ejaculate after Salmate DHA+ treatment.

The fish oil supplement, Salmate DHA+, was shown to decrease normal apoptosis during spermatogenesis in the boar. The results were an increase in sperm/ejaculate and daily sperm production.

Graphical Abstract

Graphical Abstract.

Introduction

Spermatogenesis is the process by which spermatogonia undergo mitosis, meiosis, and then morphological changes in spermiogenesis to form elongated spermatids, which travel through the epididymis to mature into spermatozoa that are ejaculated. In the boar, each type A spermatogonium can theoretically give rise to 256 spermatozoa (Parrish et al., 2017). However, from 70% to 90% of these cells are lost due to apoptosis in either mitosis or meiosis of spermatogenesis (França et al., 2005; Costa et al., 2013; Parrish et al., 2017). These are normal losses in spermatogenesis of mammals (Sharma et al., 2014).

The sperm production of males has been increased by supplementation with fish oil extracts. Fish oil has been found to increase sperm production in men with infertility due to oligospermia (Esmaeili et al., 2015). Fish oil supplementation has also been found to increase sperm production in rodents and humans who have increased apoptosis during spermatogenesis due to being given anticancer drugs (Uygur et al., 2014). Modest impacts on boar sperm production have been seen with some fish oil supplements, but palatability is a potential problem (Parsley et al., 2021). Despite the effects of fish oil on sperm production, no mechanism has been demonstrated in normal males. We speculate that fish oil may have the ability to inhibit some of the normal apoptosis that occurs during spermatogenesis. Importantly, while the process of apoptosis does remove damaged spermatogonia, primary spermatocytes, secondary spermatocytes, and round spermatids (RS), it is not clear if all the cells that become apoptotic are damaged.

While sperm quality and yield during spermatogenesis of boars are influenced by multiple factors, one major factor comes through the nutritional diet (Parsley et al., 2021). Lack of minerals, vitamins, and amino acids can contribute to lower sperm quality and yield. The requirements for essential fatty acids have not been clearly defined. Omega-3 long-chain polyunsaturated fatty acids (PUFA) have many beneficial roles in animals, including better inflammatory response, muscle contraction, wound healing, sperm concentration, semen quality, including motility and sperm membrane integrity, and antioxidant capacity of semen (Swanson et al., 2012; Yuan et al., 2023). We have been interested in the role of the omega-3 PUFAs, DHA, and EPA in boar spermatogenesis and semen. The omega-3 PUFAs of DHA and EPA are found in high amounts in fish oil. Studies done in boars found that DHA in ejaculated sperm was mostly in the tail of sperm cells (Ngcobo et al., 2021). In a further study, it was found that DHA supplementation of boars with a fish oil product was linked to a higher sperm output (Parsley et al., 2021). Interestingly, it was also found that fish oil supplementation reduced the percentage of rejected ejaculates to be processed for use in artificial insemination. This is a sign of decreased sperm damage during spermatogenesis and/or epididymal maturation. These are all signs of increased reproductive performance of boars given fish oil supplementation.

The purpose of this study was to evaluate the sperm yield and apoptosis during spermatogenesis in boars fed a control diet for 124 d or the control diet plus 26 g of Salmate DHA+ per boar for the last 62 d. The 26 g of Salmate DHA+ have 1.82 g DHA and 0.78 g EPA. We theorize that boars that consume Salmate+ will have improved sperm production compared to boars that do not consume Salmate DHA+ through changes to apoptosis during spermatogenesis.

Materials and Methods

Boars and treatments

The experimental animals consisted of 10 mature Duroc boars of a PIC terminal line that were between 438 and 686 d of age located at a commercial boar stud, the Synergy stud in West Manchester, OH. The commercial boars were already part of the boar stud’s lineup of males before the start of the trial and continued as such until harvest, so no IACUC was sought as the stud simply supplied data and tissue samples following boar harvest. All boars were first fed a control diet of corn, soybean meal, 5% fiber, plus vitamins and minerals per NRC (2012) requirements for 62 d in what will be referred to as the “before period”. This diet is considered to have negligible DHA or EPA. Following the before period, there was a second period referred to as the “after period.” During the after period, 5 boars continued the control diet while the other 5 boars were fed the control diet plus Salmate DHA+. The after period continued for 62 d. The 62-d length of time was to make sure the treatments were applied for the entire length of boar spermatogenesis (Parrish et al., 2017). Boars were randomly assigned to either the control or treated groups. The Salmate DHA+ was supplied by The Ballard Group Inc., Cincinnati, OH, and the supplemented diet had 26 g Salmate DHA+ per day per head added to the control diet. The result of adding the Salmate DHA+ to the control diet was that these boars received 1.82 g DHA and 0.78 g EPA per day during the after period. The dosage of DHA and EPA is based on a previous study (Parsley et al., 2021). Boars were in the stud from at least January 4, 2021, until shipment for harvest on January 9, 2022. The 62-d before period started on September 3, 2021, and continued until November 4, 2021. The 62-d after period began on November 5, 2021, and continued until January 7, 2022. Boars were shipped for harvest on January 9, 2022, and harvested on January 10, 2022. Harvest was done at a commercial abattoir with electrical stunning.

Semen collection and evaluation

During the trial, semen was collected from boars using the gloved hand technique as semen needed to be shipped for breeding doses. The collection frequency was between 1 and 2 times every other week and was based on the need for artificial insemination in pooled semen doses. The boar stud provided the date of collection, total sperm collected, sperm motility, and the age of individual boars. Computer-aided semen analysis was used to collect the semen data. For the study, we only evaluated semen collection data for the before period and then from days 22 to 62 in the after period. Over both the before and after periods evaluated, there were 107 ejaculates collected with data provided by the stud. All semen samples passed required values for semen shipment, which were morphological defects (proximal and distal cytoplasmic droplets) could not exceed 25%, total motility >80%, progressive motility >70%, and a minimum of 5 semen doses (2.25 × 10⁹ sperm/dose) per ejaculate.

Histology

At harvest, testes were collected with the parietal tunica vaginalis and epididymis removed, placed in separate plastic bags, labeled with boar ID and testis side, followed by covering each bag with ice. Within 1 h of harvest, each testis was then weighed.

The weight of the testicular parenchyma was determined by subtracting 6% of the testis weight from the total testis weight. The tunica albuginea was not removed when the testes were harvested and is averaged to be 6% of the testis weight in domestic animals (França and Russell, 1998). The testes were then sliced manually with a scalpel lengthwise and 4 cross sections of tissue of approximately 1 cm by 1 cm removed, with each from a different area of a teste’s parenchyma placed in Karnovky’s fixative (Karnovsky, 1965) for an initial 6 to 8 h. This step was repeated on the other testis within an animal so that both testes of the same boar were evaluated. Tissue sections were then removed from the fixative and further sliced into 2 by 4 mm cubes, refixed for an addition 24 h in new Karnovky’s fixative, rinsed with 70% ethanol twice and then stored in 70% ethanol at 4 °C. Tissue cubes were shipped in 70% ethanol to Laudier Histology, P.O. Box 78, New York, NY 10025. The commercial service of Laudier Histology embedded tissue in acrylic resin, cut blocks of the acrylic resin into 2 µm thick sections, mounted them on slides, stained with Toluidine Blue, added permount and covered with a coverslip. The service then digitally scanned 4 stained slides per sample at 400×, and this was used to quantify testis histology. All images were viewed with NDP.view2 (Hamamatsu Photonics, USA). This allowed the images to be digitally viewed at between 10× and 400× magnification with size calibration. The NDP.view2 software was used to identify stage 1 tubules with arrows, measure diameters of individual cells, outline tubules, and measure the area of tubules in µm². Images were exported and opened in ImageJ (Rasband, W.S.; NIH, Bethesda, Maryland), using the cell counter macro. This allowed the marking of which cells were being counted and the totals to be recorded.

Statistical analysis

Statistical analysis was done using SAS 9.4 (SAS Institute, Cary, NC, USA). Proc GLM was used to construct a general linear model for semen quality that included treatment (control, Salmate DHA+), period (before, after), treatment X period interaction, age of the boar at start of Salmate DHA+ feeding, and period of rest since last collection. The boar factor was considered random with other effects as fixed. The age of the boar and the period of rest since the last collection were covariates. The least square means (lsmeans) were then compared in treatment X period interaction with a slice by treatment command to produce the statistical comparisons within treatment by period. This approach was used for total sperm in the ejaculate, total motility, and progressive motility. Significance was defined as P < 0.05 and a trend when P < 0.10 but >0.05. The approach allowed accounting for any age or period of rest effects. The histology data also used Proc Glm but with a one-way analysis of variance used for round sperm/Sertoli cell (SC) and DSP. Means for each bull were used for a total of 5 bulls/treatment.

Results

Semen data

Boars were on the study diets for a total of 124 d with 62 d in the before period and 62 d in the after period. Semen data were collected in the before period (62 d) and from days 22 to 62 in the after period. Semen data were recorded for each week of the study by boar. If more than 1 ejaculate per week was collected from a boar, then data for that week were pooled. Not every boar was collected in every week of the study. The semen data included total sperm collected in each ejaculate, percentage total motile sperm (total motility), and percentage progressive motile sperm (progressive motility) although the boar stud also examined morphology as the number of cytoplasmic droplets. It is important to note that there were no discarded ejaculates in the study due to semen not meeting quality control standards. There were no effects of period of rest for total sperm per ejaculate, total motility, or progressive motility (P > 0.05). There were significant effects of age for all parameters (P < 0.05). There was no difference in the total motility within diets between the before and after period for the control diet (83 ± 1 vs. 84 ± 2; P > 0.05) or the Salmate DHA+ diet (86 ± 1 vs. 87 ± 1; P > 0.05). There was also no difference in the progressive motility for the control diet within periods (76 ± 2 vs. 77 ± 2; P > 0.05). However, there was an increase in progressive motility for the Salmate DHA+ diet in the after period (77 ± 1 vs. 82 ± 2; P < 0.05). The results for total sperm per ejaculate are shown in Figure 1. There was no difference between the before vs. after periods in the total sperm per ejaculate ×10⁹ for the control diet boars (106 ± 7 vs. 112 ± 10; P > 0.05) but there was an increase for the Salmate DHA+ diet boars in the after period (103 ± 5 vs. 123 ± 7; P < 0.05).

Figure 1.

Effect of Salmate DHA+ on sperm production. There were 107 ejaculates evaluated for this study, with 5 control and 5 Salmate DHA+ boars. Comparisons were between periods within diet (control before vs. control after; Salmate before vs. Salmate after). There were main effects of age (P < 0.05) but not days of rest (P > 0.05) between ejaculates. Abbreviations used for statistical analysis were NS, P > 0.05; *P < 0.05.

Histological evaluation of testis sections

To evaluate the impact of Salmate DHA+ on apoptosis during spermatogenesis, boars were harvested at the end of the trial and testes were processed as described in the methods. Following the approach of Swierstra (1968), Berndtson (1977), and Melo et al. (2010), we first needed to determine which seminiferous tubules in the cross sections were in stage 1. Stage 1 tubules have an absence of spermatozoa in the lumen and no elongated spermatids as shown in Figure 2. The number of RS and SC was then counted in 10 stage 1 seminiferous tubules for each boar. The RS were identified by size and shape of nuclei and SC by nucleoli (Swierstra, 1968). This number was adjusted with the modified Abercombie’s adjustment factor (Amann and Almquist, 1962; Berndston, 1977) that was calculated from the diameter of 10 RS nuclei and 10 SC nucleoli per each of 14 boars (total of 140 cells for each type), and the overall means obtained. The equation for the adjustment factor is as follows:

Figure 2.

Stage 1 of spermatogenesis. A cross-section of the testis stained with Toluidine Blue and embedded in acrylic resin. Arrows show cell types in this seminiferous tubule. Stage 1 tubules were identified by the lack of elongated spermatids or spermatozoa in the lumen. In testis morphometry, only stage 1 tubules were used for counts of SC and RS.

$${\displaystyle \begin{array}{l}{\displaystyle cellcount\times \frac{sectionthickness}{sectionthickness+\sqrt{{\left(\frac{nucleusdiameter}{2}\right)}^2-{\left(\frac{nucleusdiameter}{4}\right)}^2}}}\end{array}}$$ (1)

The coefficients for the correction factor were 0.418 (SC) and 0.267 (RS). The adjustment accounts for the diameter of the specific cell nucleoli or nuclei and the thickness of the histology section. There was also a total of 1,436 SC and 28,945 RS counted in this study, all in stage 1 seminiferous tubules. The RS/SC results are shown in Table 1. There was an increase in the RS/SC from the control boars when boars were fed Salmate DHA+ (P < 0.01). This indicates that there was a decrease in apoptosis when boars were fed Salmate DHA+.

Table 1.

Histological data for evaluation of apoptosis

Treatment	RS/SC (mean ± SEM)1
Control (boars = 5)	9.46 ± 0.84
Salmate DHA+ (boars = 5)	15.03 ± 0.97**

¹The number of RS and SC was counted in 10 stage 1 seminiferous tubules for each boar as shown in Figure 2. This number was adjusted with Abercombie’s adjustment factor and the overall means obtained. There was also a total of 1436 SC, and 28,945 RS counted in this study, all in stage 1 seminiferous tubules. A

^**indicated a difference from the control of P < 0.01.

It is possible to determine the Daily Sperm Production (DSP) from histology data. We needed to first determine the volume density using the procedure of Swierstra (1968) and Melo et al. (2010). A grid of 2,475 points was digitally placed over each of 15 images for each boar. Fifteen images per boar were used for determining the volume of the seminiferous tubules using 10× magnification. Each point was counted as either in the interstitial space or seminiferous tubule. The method for obtaining volume of the seminiferous tubules with a grid in Image J is shown in Figure 3. Seminiferous tubule volume was calculated as follows in equation 2 and 3:

Figure 3.

Grid points on testicular parenchyma (>2,400/boar). The random digital grid points (crosses) were overlaid on images and used to determine the % of parenchyma that was seminiferous tubule or interstitial tissue. This calculation was then used in the DSP shown in Table 2. The green arrow is a stage 1 seminiferous tubule.

$${\displaystyle \begin{array}{lll}{\displaystyle parenchymavolume}& =parenchymaweight& =testisweight-(.06\times testisweight)\end{array}}$$ (2)

$${\displaystyle \begin{array}{l}{\displaystyle tubulevolume=parenchymavolume\times tubule\mathrm{\%}}\end{array}}$$ (3)

Since testis density, in mammals, is situated at about 1, the testis weight in g was considered equal to its volume in ml (Melo et al., 2010). The area of stage 1 tubules was determined from outlining 10 stage 1 tubules/boar and determining the area in microns² from the calibrated images. Upon determination of seminiferous tubule volume, the length of seminiferous tubules was calculated using equation 4:

$${\displaystyle \begin{array}{l}{\displaystyle length=\frac{tubulevolume}{stage1tubulearea}}\end{array}}$$ (4)

DSP was calculated as shown in equation 5 utilizing average RS count in stage 1, and a cycle length of 8.6 d for the boar (Swierstra, 1968):

$${\displaystyle \begin{array}{l}{\displaystyle DSP=\frac{volumeoftubules\times roundspermatidcount}{durationofcycle\times areaoftubules\times sectionthickness}}\end{array}}$$ (5)

The results for DSP production are shown in Table 2. There was a trend (P = 0.067) for DSP to increase when boars were fed Salmate DHA+. The percentage of testis tissue that was seminiferous tubules did not differ between diets (P > 0.05) with an overall mean of 60% ± 3.

Table 2.

Histological data for DSP

Treatment	Sperm/g testis ± SEM1
Control	30 × 106 ± 4.5
Salmate DHA+	40 × 106 ± 1.1
Statistical difference	P = 0.067

¹Only stage 1 seminiferous tubules were included in the data analysis. A cycle length of 8.6 d was from Swierstra [11]. DSP = (volume of Seminiferous Tubules × Round spermatid count)/(duration of one cycle × area of stage 1 tubules × section thickness).

Discussion

The total number of sperm per ejaculate increased from 103 to 122 × 10⁹ on days 22 to 61 after the start of Salmate DHA+ feeding (P < 0.05) or a 1.18-fold increase. During the same interval of time there was no change in sperm per ejaculate in the boars fed the control diet. This was equivalent to 6 additional insemination doses per ejaculate if 3 × 10⁹ sperm are included in each insemination dose. Further, there was no change in total motility due to feeding Salmate DHA+ (P > 0.05) but an increase in the progressive motility (P < 0.05). Thus, there was an increase in the number of sperm ejaculated and the quality of that semen was essentially unchanged.

A unique feature of the study was that we evaluated the effects of semen production within the same boars in the before and after periods within the control (N = 5) or Salmate DHA+ (N = 5) diets. Additionally, we waited in the after period until 21 d had passed. This was to allow spermatozoa, elongated spermatids, and late-stage RS to complete spermatogenesis and be ejaculated. As the study was designed to look at apoptosis in spermatogenesis (França et al., 2005; Costa et al., 2013; Parrish et al., 2017), the 21-d period was needed to see the impact of Salmate DHA+ on mitosis and meiosis of spermatogenesis with a change in sperm within the ejaculate.

Apoptosis during spermatogenesis was directly evaluated from testis morphometry in stage 1 seminiferous tubules and we found that the number of RS/SC increased in Salmate DHA+ fed boars (p < 0.01). If a spermatogonia gave rise to the theoretical maximum number of RS, then there would be 256 RS per SC (França et al., 2005; Costa et al., 2013; Parrish et al., 2017). Through apoptosis however 70% and 90% of potential RS are lost as a normal part of spermatogenesis. These results support the hypothesis that feeding a fish oil supplement in the form of Salmate DHA+ decreased apoptosis. This is the first study to demonstrate a mechanism of fish oil in increasing sperm production in a normal mammalian male.

Other features of testis morphometry were that we found that the percentage of testis that was seminiferous tubules was 60 ± 3% which is lower than the 83% and 85% found by França et al. (2005). This may be due to these boars being mature and between 438 and 686 d of age when the Salmate DHA+ feeding began or the breed of boars, which was Duroc. The RS/SC ratio for control-fed boars was 9.46 ± 0.84 and compares to the 12.4 seen by França et al. (2005). It is unclear why our control-fed boars have fewer RS/SC or efficiency of spermatogenesis, but this again may be due to the age of boars and/or breed.

From testis morphometry we also determined the DSP. There was a trend (P = 0.067) for an increase in DSP from 30 × 10⁶ to 40 × 10⁶ sperm/g testis or a 1.33-fold increase. While more than the increase due to Salmate DHA+ feeding seen with direct measurement of sperm per ejaculate (1.18-fold increase), the testis morphometry was done at the end of the study and thus there was more time for the impact of Salmate DHA+. The DSP compares to 24 to 27 × 10⁶ sperm/g testis as seen by França et al. (2005). The results of the direct measurement of sperm apoptosis due to Salmate DHA+ feeding was done as the ratio of RS/SC (1.59-fold increase), sperm/ejaculate (1.18-fold increase), and histologically measured DSP (1.33-fold increase). All agree in that Salmate DHA+ feeding results in an increase in sperm output. In this trial, there was no palatability issue with Salmate DHA+ feeding that has been seen with other fish oil products (Parsley et al., 2021).

Other studies have seen that sperm production is increased due to fish oil supplementation in rodents and humans but that was in males that have been given anticancer drugs that induce apoptosis or in males with oligospermia who are known to have increased apoptosis already (Uygur et al., 2014; Esmaeili et al., 2015). In boars, the results of fish oil feeding have been mixed. Increases in sperm output were observed by Parsley et al. (2021) and Estienne et al. (2008)  but not by Yeste et al. (2011) or Rooke et al. (2001) or Castellano et al. (2010).

Studies in boars must consider seasonal effects on sperm production. In the boar, reduced total sperm are produced due to summer heat stress (Smital, 2009; Parrish et al., 2017). As summer effects can continue to still impact sperm production in November when heat stress ended in September in Wisconsin (Parrish et al., 2017), it is important to measure sperm production within boars and be sure both control and treated boars are exposed to the same environmental conditions over time. This was done in this study and found RS/SC to increase when boars are fed Salmate DHA+.

In this study, the boars used were mature PIC line 800 Duroc boars. Interestingly in a comparison of boar breeds, Durocs produced more sperm than either Large-White or Pietrain (Yeste et al., 2011). The breed of boar may have impacted some of the parameters observed when compared to that of França et al. (2005), for example, but the data were clear in that Salmate DHA+ feeding decreased apoptosis and increase sperm output.

The fish oil supplement fed in this study was Salmate DHA+ that has EPA and DHA. Further, it is a refined fish oil, vitamin E, homogenized into 100 µm droplets and coated with hydrophobic starch. The result is a product that resists oxidation of the PUFA and the starch helps with intestinal release of the PUFA and prevents palatability problems that can occur with fish oil oxidation (Parsley et al, 2021).

We did not attempt to determine the minimum or maximum amounts of Salmate DHA+ that would change apoptosis in spermatogenesis. However, there are data to demonstrate that if you inhibit apoptosis in knockout models for BAX-deficient mice, there is an increase in spermatogonia and primary spermatocytes, a thickening of the basal compartment in the seminiferous tubule and then degeneration of primary spermatocytes, secondary spermatocytes, and RS through an apoptosis-independent mechanism (Russell et al., 2002). This then leads to sterility in these mice. The normal relationship of RS per SC is massively disrupted. In previous studies with mixed results with feeding of a fish oil supplement, we cannot comment about the level fed except that sometimes an increase in sperm output and/or quality is seen but not in others (Rooke et al., 2001; Estienne et al., 2008; Yeste et al., 2011; Parsley et al., 2021). Caution is warranted about potential overfeeding of a fish oil supplement with DHA and EPA that could cause results similar to that in BAX-deficient mice (Russell et al., 2002).

Conclusions

The study demonstrates that feeding Salmate DHA+ to boars as a top dress at 26 g per day per head increased total sperm per ejaculate, progressive motility, and decreased apoptosis in spermatogenesis. The 26 g Salmate DHA+ contained 1.82 g DHA and 0.78 g EPA. Salmate DHA+ fed to boars did not present any palatability issues. This is the first study to demonstrate that the effects of a fish oil supplement impact sperm production by decreasing apoptosis during spermatogenesis.

Glossary

Abbreviations

CASA

computer aided semen analysis

DHA

docosahexaenoic acid

EPA

eicosatetraenoic acid

lsmeans

least square means

NRC

National Research Council

PUFA

polyunsaturated fatty acid

RS

round spermatid

SC

Sertoli cell

PIC

Pig Improvement Company

µm

micron

Author Contributions

John J. Parrish (Conceptualization, Formal analysis, Investigation, Methodology, Project administration, Resources, Validation, Visualization, Writing—original draft, Writing—review & editing), Tom J. Gall (Conceptualization, Investigation, Methodology, Project administration, Resources, Writing—review & editing), Mark E. Wilson (Conceptualization, Methodology, Project administration, Writing—review & editing), Mike A. Parsley (Resources), and Mark R.M. Ballard (Funding acquisition, Resources)

Conflict of interest statement. M.E.W. and T.J.G. are consultants to Feedworks USA, LTD. M.A.P. markets Salmate DHA+. M.R.M.B.’s company manufactures Salmate DHA+ and was the funder for the project. All were involved in designing experiments but only T.J.G. was involved in collection of data. T.J.G. and M.E.W. were involved with interpretation of the data. The funder had no role in the decision to publish results. J.J.P. was supplied with access to the boars and testes upon harvest along with funds to process testes section for histology and cost of meeting attendance.

Data Availability

Data will be made available upon request of the corresponding author: John Parrish, jjparris@wisc.edu.