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Microelements in seminal and serum plasma are associated with fresh semen quality in Yorkshire boars
Theriogenology 132 (2019) 88e94
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Microelements in seminal and serum plasma are associated with fresh semen quality in Yorkshire boars Yinghui Wu a, Liangliang Guo a, Zihui Liu b, Hongkui Wei a, Yuanfei Zhou a, Jiajian Tan c, Haiqing Sun c, Shengqing Li b, Siwen Jiang d, e, *, Jian Peng a, e, ** a
Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China Department of Chemistry, College of Science, Huazhong Agricultural University, Wuhan, 430070, PR China YangXiang Joint Stock Company, Guigang, 537000, PR China d Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, PR China e The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2018 Received in revised form 2 April 2019 Accepted 3 April 2019 Available online 6 April 2019
This study aimed to explore associations between semen quality and trace element level in serum and seminal plasma in Yorkshire boars. Semen quality of 112 Yorkshire boars was assessed for 13 weeks to calculate semen utilization rate, which was then divided into three categories: low utilization rate group (LG, < 60% utilization rate), medium utilization rate group (MG, 60e80%), and high utilization rate group (HG, > 80%). After grouping, serum and seminal plasma samples of selected boars were collected to determine concentrations of 10 elements including Ca, Mg, Cu, Fe, Zn, Mn, Se, Cr, Pb and Cd using inductively coupled plasma mass spectrometry. Results showed the increase of semen utilization rate was accompanied by the increase of sperm motility and the decrease of abnormal sperm rate among three groups (P < 0.01). Serum Fe concentration in LG boars was lower than that in HG boars (P < 0.05). Regression analysis revealed serum Fe concentration was positively correlated with sperm motility (r ¼ 0.251; P < 0.05), while negatively correlated with abnormal sperm rate (r ¼ 0.207; P < 0.05). However, MG and HG boars had lower serum Se concentration than LG boars (P < 0.05), and serum Se concentration contribution to sperm motility varied in a quadratic manner (Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300; P < 0.01). Semen utilization rate tended to decrease with the increase of seminal plasma Pb concentration (P ¼ 0.09). Regression analysis exhibited seminal plasma Pb negatively related to sperm motility (r ¼ 0.237; P < 0.05), while positively correlated with abnormal sperm rate (r ¼ 0.237; P < 0.05). Furthermore, seminal plasma Pb was the most influential factor among trace element in serum and seminal plasma on sperm motility basing on the generalized linear model analysis (P < 0.05). Sperm motility decreased by approximately 3.47% when seminal plasma Pb concentration increased from 0 mg/L to 11.16 mg/L. In conclusion, deficiency of serum Fe reduces semen utilization rate by impairing sperm motility and morphology, whereas excessive serum Se decreases sperm motility. More importantly, the mere existence of seminal plasma Pb has more impact on semen quality than other trace elements in serum and seminal plasma in Yorkshire boars. © 2019 Published by Elsevier Inc.
Keywords: Semen quality Seminal plasma Serum Microelements Yorkshire boar
1. Introduction * Corresponding author. Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, PR China. ** Corresponding author. Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China. E-mail addresses: [email protected] (S. Jiang), [email protected]. edu.cn (J. Peng). https://doi.org/10.1016/j.theriogenology.2019.04.002 0093-691X/© 2019 Published by Elsevier Inc.
Essential trace elements serve as structural components of body tissues, constituents of body fluids and various enzymes in major metabolic pathways and are indispensable for physiological reproductive function [1]. However, the accumulation of nonessential potentially toxic trace elements in the body also adversely affects mammalian male reproductive system [2e4]. Therefore, maintaining reasonable addition of essential trace elements and
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avoiding toxic trace elements are important for male reproductive system. Trace elements of Cu, Fe, Zn, and Se play a key role on metabolism of carbohydrates, proteins and lipids and they have equal, beneficial or detrimental effects, depending on their balance, on mammalian male reproductive functions [5e8]. A lower level of Cu enhances sperm membrane lipid peroxidation and sperm forward motility, but excess of Cu causes marked inhibition of these biochemical parameters [9]. Seminal plasma Fe relates to sperm progressive motility, gross motility and viability in water buffalo [6]. In regard to Se, it acts as an antioxidant through various selenoproteins viz. glutathione peroxidase, thioredoxin reductase and selenoprotein P. Se deficiency and to a lesser extent Se excess both lead to decreased expression pattern of cJun and cFos genes in testicular germ cells which might be responsible for decreased germ cell number, reduced fertility, and disrupted spermatogenesis [10]. Pb and Cd, which is pervasive in environment and accumulates in the body during lifetime (especially Pb) are toxic elements for humans and other mammals [11]. The presence of Pb and Cd in seminal plasma decrease sperm motility by increasing free radical formation as well as antioxidants damage [12,13]. Although trace element test in seminal plasma is frequently used because of the importance for sperm metabolism, function and survival, that in serum is also an important indicator of testicular germ cells function and spermatogenesis in mammalian males [8,14]. However, few studies were conducted to combine trace element test in both serum and seminal plasma for semen quality analysis. In addition, it is reported the absorption and metabolism of trace elements are different among cow breeds [15]. Our previous study has demonstrated serum Cu, Fe, Mn and seminal plasma Cd levels affect semen quality of Duroc boars [16], but relationship between trace element and semen quality in other boar breeds is not clear. We hypothesized trace elements in serum and seminal plasma both can be marks of semen quality, and relationships between trace element and semen quality in Yorkshire boars are different from that in Duroc boars. Therefore, the objectives of this study were to: 1) test trace elements levels (Ca, Mg, Cu, Fe, Zn, Mn, Se, Cr, Pb, and Cd) in serum and seminal plasma; 2) analyze relationships between trace elements and semen quality using regression model; 3) determine the most influential factor among trace element in serum and seminal plasma on semen quality using generalized linear model (GLM) in Yorkshire boars. 2. Materials and methods All animal handling protocols were approved by the Animal Care and Use Committee of the College of Animal Science and Technology, Huazhong Agricultural University (Committee of Science and Technology). 2.1. Study population To evaluate the relationships between trace element level in serum and seminal plasma and semen quality, 112 healthy Yorkshire boars aged from 9 to 42 mo were selected at Yaji Mountain AI Center of YangXiang Joint Stock Company, Guangxi province, China. The Yorkshire boars were raised in stalls (0.79 by 2.40 m), which were fully slatted with concrete slats (90 mm solid width, 23 mm slots). The ambient temperature inside the house was maintained at around 19.8 Ce25.4 C and extra light was provided when the natural photoperiod could not meet the daylight regime of 12 h of lighting. The Yorkshire boars were restricted to 2.5 kg/d of commercially feed, which was the same as the diet in our previous study [16]. In addition, The Yorkshire boars were provided access to water ad libitum.
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2.2. Semen quality assessment and experimental grouping of boars Semen sample was collected by the gloved-hand technique with a frequency of three times every two weeks during experiment period. For semen collection, sperm-rich ejaculate fractions were collected and the gelatinous fraction was strained from the ejaculate through 4 layers of cotton-mesh gauze. After sampling, four basic semen parameters were examined. Briefly, semen volume was measured by weighing each ejaculate and considering 1 g of semen to be equal to 1 mL. Sperm concentration was tested three times for each semen sample by using a sperm density meter (Fujihira Industry Co., LTD, Tokyo, Japan) and the average value was considered as the final concentration. To determine sperm motility, a computer-aided sperm analysis system (CASA system, MLe210JZ, Nanning SongJinTianLun Biological Technology Co., LTD, Nanning, China) was used. The CASA system was loaded with the MAILANG sperm automatic analytical software, and conjugate high-contrast sperm optical imaging lighting device and MAILANG computer sperm scanning counting board. The ejaculates were diluted (1:1) immediately with the isothermal Beltsville Thawing Solution (Jin Li Livestock Equipment Co., LTD, Wuhan, China). A 10-mL aliquot of semen sample was placed in a pre-warmed (37 C) microscope slide and covered with a cover glass. The grey level was set between 30 and 50, and minimum and maximum diameters were set between 3 and 20 mm in black mode when sperm motility was analyzed for five fields. To examine sperm morphology, the reagents were prepared by dissolving 0.67 g of eosin Y and 0.9 g of NaCl in 100 ml of purified water and adding 10 g of nigrosin the 100 ml of eosin Y solution firstly. Then a 10 mL aliquot of semen with an equal volume of eosinenigrosin suspension was mixed and a smear on a glass slide was made for air drying. Finally, at least 200 spermatozoa was evaluated in each replicate for each slide at a magnification of 400 under bright field optical microscope in order to achieve an acceptably [17]. Sperm with head defects, neck and mid-piece defects, tail defects, and excess residual cytoplasm were considered unnormal in this study. Total sperm number (volume concentration) and functional sperm number [total sperm number motility (1 e abnormal sperm rate)] were calculated according to Smital et al. (2004) and Wolf and Smital (2009) [18,19]. Boar semen with agglutinated sperm, sperm motility lesser than 70%, or abnormal sperm rate higher than 20% was considered as not available based on the cut-off values recommended by previous studies (normal sperm morphology 80% and sperm motility 70%) [16,20]. Therefore, the border between the nontoxic or toxic effect of elements on sperm motility and abnormal sperm rate were set at sperm motility of 70% and abnormal sperm rate of 20%, respectively. Semen utilization rate was calculated basing on semen quality of each ejaculate. According to semen utilization rate, 112 Yorkshire boars were allocated into three groups: low utilization rate group (LG, < 60% utilization rate, n ¼ 16), medium utilization rate group (MG, 60e80%, n ¼ 28), and high utilization rate group (HG, > 80%, n ¼ 68). 2.3. Sample collection and element concentrations analysis Serum and seminal plasma samples from 112 Yorkshire boars were collected in September 2016 after the semen quality of each boar was determined. Blood samples were collected by venipuncture after an overnight fast of at least 12 h, blood was allowed to clot and serum was recovered after centrifugation at 1500g for 10 min at room temperature. Sperm samples were collected at the same time as blood samples, and further centrifugation was performed to obtain seminal plasma samples. Serum and seminal plasma samples were stored at 80 C until trace element analysis.
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In addition, diet and drinking water samples of boars were also collected and stored at 4 C for trace element analysis. The method for determining the content of serum and seminal plasma elements were described in our previous study [16]. Briefly, frozen serum and seminal plasma samples were liquefied and the samples were digested in 10 mL polyethylene pipe in a fume cupboard with a 1:2 solution of highly purified HNO3 under moderate heating conditions (80 C) for about 4 h until the solution was transparent. The samples were diluted with Milli-Q water after cooling to room temperature, then these samples were mixed and filtered for determination of Ca, Mg, Cu, Fe, Zn, Mn, Se, Pb, and Cd. In addition, diet sample was pretreated by wet digestion. Drinking water sample was filtered and directly detected for element concentration. The element content in the sample was detected by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900, Agilent Technologies, Tokyo, Japan). Argon was used as the carrier gas and the isotopes 44Ca, 24Mg, 63Cu, 56Fe, 64Zn, 55Mn, 78Se, 52 Cr, 208Pb, and 111Cd were selected as analytical masses in the ICPeMS normal sensitivity mode. A Collision Reaction Interface was used for the measurements of Ca, Fe and Se to reduce common polyatomic interferences. 2.4. Statistical analysis All analyses in this study were completed in SAS (version 9.4; SAS Inst. Inc., Cary, NC). Firstly, ShapiroeWilk's test was used to verify normality of semen traits and element concentrations. Squareeroot or natural log (ln) transformed was used to correct when these parameters were not normal distribution. Indicators such as sperm motility, abnormal sperm rate, and serum Mn concentration still showed skewed distributions (nonenormal) after data transformed. Therefore, results were presented as mean ± SD for parameters with normal distribution and median for data with skewed distribution in the present study. Secondly, significant differences in semen traits and element concentrations among three groups were tested using oneeway ANOVA (normal distribution) or KruskaleWallis (skewed distribution). Thirdly, linear regression model and nonlinear regression model were carried out to explore the quantitative relationship between semen quality and element concentrations in serum and seminal plasma. Finally, the GLM, which considered semen traits as dependent variables and
element concentrations as independent variables was performed to explore the most influential factor among trace elements in serum and seminal plasma on semen quality. Briefly, element concentrations were divided into five groups in an increasing order and analyzed as classified variables. Sample sizes of the five groups were 22, 22, 22, 23, and 23, respectively. The equations of sperm motility (model 1) and abnormal sperm rate (model 2) were presented as follows. The significance level was set at 5% in all statistical analyses. Yklmn¼m1þBk þ Tl þ Sm þ Mn þ eklmn
(model 1)
Yln¼m1þTl þ Mn þ eln
(model 2)
where the Yklmn (model 1) was the value of the given sperm motility for the kth serum Cu, lth serum Fe, mth serum Se, and nth seminal plasma Pb; the Yln (model 2) was the value of the given abnormal sperm rate for the lth serum Fe and nth seminal plasma Pb; Bk is the effect of the kth serum Cu (k ¼ 1, 2, 3, 4, 5); Tl is the effect of the lth serum Fe (l ¼ 1, 2, 3, 4, 5); Sm is the effect of the mth serum Se (m ¼ 1, 2, 3, 4, 5); Mn is the effect of the nth seminal plasma Pb (n ¼ 1, 2, 3, 4, 5); and eklmn and eln were the residual effects of model 1 and model 2, respectively. 3. Results Table 1 shows that the contents of Cu, Fe, Zn, Mn, Se and Cr in diet were 17.78 mg/kg, 271.63 mg/kg, 295.71 mg/kg, 83.36 mg/kg, 0.32 mg/kg, and 1.61 mg/kg, respectively. Toxic elements of Pb and Cd were observed in diet and their concentrations were 0.14 mg/kg and 0.03 mg/kg, respectively. However, these two toxic elements were not found in drinking water. Semen quality parameters and serum and seminal plasma element concentrations of Yorkshire boars in different semen utilization groups are presented in Table 2 and Table 3. LG boars had lower functional sperm number, lower sperm motility and higher abnormal sperm rate than MG boars and HG boars (P < 0.01), and MG boars had lower sperm motility and higher abnormal sperm rate than HG boars (P < 0.01). Furthermore, as compared to HG boars, LG boars had lower serum Fe concentration (P < 0.05) and higher serum Se concentration (P < 0.05). Semen utilization rate
Table 1 Concentration of elements in diet and drinking water of boars.
Diet, mg/kg Drinking water, mg/L a
Ca
Mg
Cu
Fe
Zn
Mn
Se
Cr
Pb
Cd
7600.03 3043.94
2069.02 1806.13
17.78 Nb
271.63 N
295.71 38.34
83.36 0.31
0.32 0.12
1.61 N
0.14 N
0.03 N
recommended amount of mineral elements for boars in NRC (2012). N: the element detection content is less than 0.
b
Table 2 Semen quality parameters of Yorkshire boars in different semen utilization groups (n ¼ 112)a.
Semen volume, mL Sperm concentration, 106 mL1 Total sperm number, 109/ejaculate Functional sperm number, 109/ ejaculate Sperm motility, % Abnormal sperm rate, % a
Low utilization group (<60%, n ¼ 16)
Medium utilization group (60%e80%, n ¼ 28)
High utilization group (>80%, n ¼ 68)
Pvalueb
226.34 ± 70.85 253.40 ± 79.96 56.58 ± 23.35 35.99 ± 19.84B
263.15 ± 71.57 277.14 ± 80.55 66.79 ± 18.46 52.05 ± 18.72A
252.36 ± 70.70 274.9 ± 62.38 67.14 ± 17.08 52.87 ± 13.72A
0.25 0.52 0.13 <0.01
77.80 [66.70e82.34]C 23.46 [18.49e32.25]A
85.00 [82.96e87.34]B 13.06 [11.79e16.77]B
87.45 [86.47e88.84]A 10.07 [8.85e11.61]C
<0.01 <0.01
The results are presented as mean ± standard deviation for normal distribution or median [quartile 25- quartile 75] for skewed distribution. Different superscripts within a row indicate significant differences. P < 0.01.
b
A,B,C
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Table 3 Serum and seminal plasma element concentrations of Yorkshire boars in different semen utilization groups (n ¼ 112).a
Serum Ca, mg/L Mg, mg/L Cu, mg/L Fe, mg/L Zn, mg/L Mn, mg/L Se, mg/L Cr, mg/L Pb, mg/L Cd, mg/L Seminal plasma Ca, mg/L Mg, mg/L Cu, mg/L Fe, mg/L Zn, mg/L Mn, mg/L Se, mg/L Cr, mg/L Pb, mg/L Cd, mg/L
Low utilization group (<60%, n ¼ 16)
Medium utilization group (60%e80%, n ¼ 28)
High utilization group (>80%, n ¼ 68)
P-valueb
123.35 ± 13.35 22.41 ± 3.00 2.57 ± 0.44 1.11 ± 0.33b 0.83 ± 0.15 5.03 [0e14.90] 229.33 ± 69.57a 148.14 [110.26e199.92] 5.50 [0e11.09] 0.10 [0e0.43]
114.06 ± 16.74 22.94 ± 4.21 2.46 ± 0.59 1.15 ± 0.30ab 0.94 ± 0.29 0.52 [0e9.51] 189.80 ± 50.26b 170.44 [118.33e202.91] 9.11 [3.52e19.56] 0.15 [0e0.40]
114.92 ± 17.90 21.44 ± 3.38 2.33 ± 0.47 1.34 ± 0.39a 0.88 ± 0.29 2.03 [0e14.84] 190.29 ± 49.63b 160.64 [104.83e200.63] 7.02 [0.24e16.59] 0.20 [0e0.52]
0.17 0.15 0.19 0.02 0.41 0.84 0.04 0.64 0.25 0.35
32.94 ± 10.44 115.15 ± 48.91 49.16 [11.60e53.52] 0.54 [0.39e0.68] 23.88 ± 14.22 5.68 [1.34e9.59] 76.97 ± 27.41 47.68 [12.13e104.86] 5.44 [0.44e10.60] 0 [0e0.49]
30.90 ± 12.61 114.05 ± 59.55 44.16 [24.48e51.72] 0.44 [0.17e0.80] 22.51 ± 14.91 7.11 [2.44e10.35] 64.46 ± 30.96 53.16 [21.76e90.00] 1.66 [0e7.65] 0 [0e0.22]
29.60 ± 12.16 136.54 ± 56.84 46.87 [17.38e56.70] 0.51 [0.28e0.78] 26.64 ± 12.72 4.83 [2.21e10.18] 71.71 ± 31.25 58.71 [23.32e86.97] 0.75 [0e4.98] 0 [0e0.32]
0.65 0.23 0.92 0.76 0.44 0.82 0.49 0.95 0.09 0.61
a
The results are presented as mean ± standard deviation for normal distribution or median [quartile 25- quartile 75] for skewed distribution. Different superscripts within a row indicate significant differences. a,b P < 0.05.
b
Fig. 1. Relationships between sperm motility and the element concentrations of Yorkshire boars (n ¼ 112). (A) Sperm motility ¼ 2.441 Cu(serum) þ91.18; r ¼ 0.207, P < 0.05. (B) Sperm motility ¼ 3.349 Fe(serum) þ81.02; r ¼ 0.251, P < 0.05. (C) Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300, P < 0.01. (D) Sperm motility ¼ 0.301 Pb(seminal) þ86.21; r ¼ 0.237, P < 0.05.
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tended to decrease with the increase of seminal plasma Pb among groups. Regression analysis results of relationships between each element and sperm motility and abnormal sperm rate are summarized in Figs. 1 and 2. As shown in Fig. 1, sperm motility was negatively correlated to serum Cu and seminal plasma Pb, while positively correlated to serum Fe (P < 0.05). A quadratic relationship that increased firstly and then decreased was observed between serum Se and sperm motility (P < 0.05). As shown in Fig. 2, abnormal sperm rate decreased with the increase of serum Fe concentration, but increased with the increase of seminal plasma Pb concentration (P < 0.05). The GLM analysis was performed to determine the most influential factor among trace element in serum and seminal plasma on sperm motility. As shown in Table 4, only seminal plasma Pb showed a significant effect on sperm motility (P < 0.05), while serum Cu, serum Fe and serum Se had no significant impact on sperm motility in the GLM analysis. As shown in Table 5, sperm
Fig. 2. Variations of abnormal sperm rate with the element concentrations in Yorkshire boars (n ¼ 112). (A) Abnormal sperm rate ¼ 3.613 Fe(serum)þ17.81; r ¼ 0.207, P < 0.05. (B) Abnormal sperm rate ¼ 0.322 Pb(seminal)þ11.94; r ¼ 0.237, P < 0.05.
motility in 80e100% seminal plasma Pb group was lower than that in 20% and 20e40% seminal plasma Pb groups (P < 0.05) and it decreased by approximately 3.47% when the seminal plasma Pb increased from 0 mg/L to 11.16 mg/L (P < 0.05). Serum Fe and seminal plasma Pb had no significantly effect on abnormal sperm rate in the GLM analysis, although these two element concentrations were associated with abnormal sperm rate in the correlation analysis. 4. Discussion The relationship between trace element in serum and seminal plasma and semen quality of Yorkshire boars was investigated in this study. Results indicated serum Fe, Se and seminal plasma Pb concentrations were different among different semen utilization groups. Regression analysis revealed serum Fe concentration positively, while excessive serum Se and seminal plasma Pb concentrations negatively affects semen quality. Furthermore, GLM analysis exhibited the level of seminal plasma Pb has a greater impact on sperm motility than other influencing elements. Trace elements are important for the maintenance of normal spermatogenesis and sperm function. Fe is involved in redox process and acts as components of many enzymes and metalloprotein compounds [21]. In this study, lower serum Fe level reduced semen utilization rate by decreasing sperm motility and increasing abnormal sperm rate, which indicated serum Fe concentration may be inadequate for boars. It has been proposed sertoli cells bind and internalize diferric transferrin from blood at their basal pole, remove Fe from the basal compartment, and provide it to the meiotic spermatocytes and differentiating spermatids [22]. Serum Fe concentration limits energy production and spermatogenesis by affecting Fe metabolism in germ cells [23]. In addition, the lack of Fe affects the activity of Feedependent or Feecontaining enzymes, which increases lipid peroxidation in boar spermatozoa [24]. Therefore, inadequate serum Fe led to poor semen quality in Yorkshire boars as our previous findings in Duroc boars [16]. Se is required for vital proteins and enzymes that has important role in spermatogenesis and male fertility [25]. However, the beneficial or detrimental effect of Se on semen quality depends on its contents in body. Pipan et al. (2017) reported Se in fresh boar seminal plasma had positive correlation with sperm quality after storage [26]. However, there was no relationship between seminal plasma Se and semen quality in our study. This result may demonstrate the seminal plasma Se level in our study (70.76 mg/L), which higher than that in Pipan's study (6.21 mg/L) was sufficient for maintaining semen quality. But interestingly, we found it existed a quadratic relationship (Fig. 1C. Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300, P < 0.01) between serum Se and semen quality in Yorkshire boars of the present study. Sperm motility improved as the serum Se level increased below the level of 170 mg/L (the vertex of parabola), with decreasing thereafter. Furthermore, when serum Se was higher than 367.50 mg/L (calculated basing on the equation), sperm motility decreased below 70%, which was considered as cut off value for poor semen quality. To summarize we could speculate that: 1) when serum Se level < 170 mg/L, sperm motility improved as the serum Se level increased; 2) when serum Se level ranged 170 mg/L to 367.50 mg/L, sperm motility decreased but without toxicity; 3) when serum Se level > 367.50 mg/L, sperm motility decreased below 70% with toxicity. We speculated the probable reason may be the pro-oxidant effect of excess Se, particularly in the form of selenite [27,28]. However, there was no significant correlation between serum Se and semen parameters in Duroc boars [16]. Difference in the relationship between serum Se and semen parameters in our previous [16] and this studies indicated the absorption and tolerance of Se may be different among different boar breeds. Pogge et al. (2011) demonstrated Simmental cattle had
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Table 4 GLM analysis the effect of multiple elements concentrations on semen quality parameters in Yorkshire boars (n ¼ 112). Parameters
Models
Results
Sperm motility
Yklm¼m1þBk þ Tl þ Sm þ Mn þ eklmn (model 1)
Abnormal sperm rate
Yln¼m1þTl þ Mn þ eln (model 2)
Source
DF
Mean square
F value
P-value
Serum Cu Serum Fe Serum Se Seminal plasma Pb Serum Fe Seminal plasma Pb
4 4 4 4 4 4
22.84 59.96 54.49 86.03 47.19 84.07
0.57 1.53 1.22 2.19 1.07 1.90
0.69 0.20 0.31 0.04 0.38 0.12
where the Yklmn (model 1) was the value of the given sperm motility for the kth serum Cu, lth serum Fe, mth serum Se, and nth seminal plasma Pb; the Yln (model 2) was the value of the given abnormal sperm rate for the lth serum Fe and nth seminal plasma Pb; Bk is the effect of the kth serum Cu (k ¼ 1, 2, 3, 4, 5); Tl is the effect of the lth serum Fe (l ¼ 1, 2, 3, 4, 5); Sm is the effect of the mth serum Se (m ¼ 1, 2, 3, 4, 5); Mn is the effect of the nth seminal plasma Pb (n ¼ 1, 2, 3, 4, 5); and eklmn and eln were the residual effects of model 1 and model 2, respectively.
Table 5 Effect of boar seminal plasma Pb on the sperm motility (model 1)a. Seminal plasma Pb
n
Concentration, mg/L
Sperm motility, %
20% 20e40% 40e60% 60e80% 80e100%
22 22 22 23 23
0 [0-0] 0 [0e0.01] 1.84 [0.75e3.48] 5.44 [4.67e7.65] 11.16 [9.60e14.74]
87.33 88.56 86.56 86.53 84.48
Statistical significance of seminal plasma Pb 20%
[86.47e89.06] [85.00e88.87] [85.53e87.94] [83.60e87.67] [81.38e87.38]
20e40%
40e60%
60e80%
80e100%
NSb
NS NS
NS NS NS
*c * NS NS
aThe results are presented as median [quartile 25- quartile 75] for skewed distribution. b NS: not significant. c * significant on the level P < 0.05.
lower plasma Se values compared to Angus cattle when calves received a same corn-silage based diet [29]. In this study, we also found serum Cu has a negative influence on sperm motility, but there was no significant difference in serum Cu among different semen utilization groups. This may be due to a slight excess of serum Cu existed in experiment boars. Olivari et al. (2009) reported high concentration serum Cu acts as a proeoxidant and leads to reactive oxygen species (ROS) production [30]. Pb and Cd are two common toxic elements for male reproductive system, and they were detected in boar diet in this study. Pb compounds initiate oxidative stress and lead to produce ROS, which causes lipid peroxidation of sperm membrane [31]. Many studies have reported total sperm number, sperm motility, sperm morphology [11,31,32], and sperm metabolism [33] are damaged by exposing to Pb. In the present study, toxic element Pb in seminal plasma showed a more significant effect on semen parameters in Yorkshire boars. This result indicated sperm exposed to toxic elements may suffer more damage than that under deficiency and excess of trace nutritional elements. Similarly, Eghbali et al. (2010) also claimed seminal plasma Pb has an adverse effect on motility and viability of spermatozoa after ejaculation in water buffalo [6]. Toxic element Cd in seminal plasma decreases sperm concentration and impairs sperm motility and morphology in human [34,35]. It possibly because Cd causes ROS production and leads to excessive protein oxidation, lipid peroxidation, and cell death [36,37]. However, only a negative correlation between seminal plasma Cd and sperm concentration was found in this study. This result of Yorkshire boars was not consistent with that reported in Duroc boars [16]. The possible reason is due to inconsistencies in absorption efficiency of toxic elements between Yorkshire boars and Duroc boars. The median of seminal plasma Cd concentration in Yorkshire boars and Duroc boars were 0 mg/L and 0.08 mg/L, respectively. We suspect Yorkshire boars are less efficient at Cd uptake or transport from blood to semen, which led to the lower level of Cd in Yorkshire boars than that in Duroc boars.
5. Conclusions In conclusion, deficiency of serum Fe affects semen quality by impairing sperm motility and morphology, while excess of serum Se is toxic to semen quality in Yorkshire boars. More importantly, toxic element Pb in seminal plasma shows a more significant effect on semen parameters than other influencing elements in Yorkshire boars. We suggest producers should pay attention to the reasonable addition of nutritional trace elements and monitor toxic elements in diet of Yorkshire boars. Conflicts of interest None of the authors has any conflict of interest to declare. Acknowledgments This research was supported financially by the National Key Research and Development Project of China (No. 2017YFD0502004), National key research and development program (No. 2018YFD0501003), Hubei Provincial Creative Team Project of Agricultural Science and Technology (No. 2007-620), and China Agriculture Research System (No. CARS-36). Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.theriogenology.2019.04.002. References [1] Mertz W. The essential trace elements. Science 1981;213:1332e8. https://doi. org/10.1126/science.7022654. [2] Ahmad SAWG. Effects of lead on the male reproductive system in mice. J Toxicol Environ Health 1999;56:513e21. https://doi.org/10.1080/ 009841099157953. [3] Monsefi M, Alaee S, Moradshahi A, Rohani L. Cadmium-induced infertility in male mice. Environ Toxicol 2010;25:94e102. https://doi.org/10.1002/tox.
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Microelements in seminal and serum plasma are associated with fresh semen quality in Yorkshire boars Yinghui Wu a, Liangliang Guo a, Zihui Liu b, Hongkui Wei a, Yuanfei Zhou a, Jiajian Tan c, Haiqing Sun c, Shengqing Li b, Siwen Jiang d, e, *, Jian Peng a, e, ** a
Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China Department of Chemistry, College of Science, Huazhong Agricultural University, Wuhan, 430070, PR China YangXiang Joint Stock Company, Guigang, 537000, PR China d Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, PR China e The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, 430070, PR China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 September 2018 Received in revised form 2 April 2019 Accepted 3 April 2019 Available online 6 April 2019
This study aimed to explore associations between semen quality and trace element level in serum and seminal plasma in Yorkshire boars. Semen quality of 112 Yorkshire boars was assessed for 13 weeks to calculate semen utilization rate, which was then divided into three categories: low utilization rate group (LG, < 60% utilization rate), medium utilization rate group (MG, 60e80%), and high utilization rate group (HG, > 80%). After grouping, serum and seminal plasma samples of selected boars were collected to determine concentrations of 10 elements including Ca, Mg, Cu, Fe, Zn, Mn, Se, Cr, Pb and Cd using inductively coupled plasma mass spectrometry. Results showed the increase of semen utilization rate was accompanied by the increase of sperm motility and the decrease of abnormal sperm rate among three groups (P < 0.01). Serum Fe concentration in LG boars was lower than that in HG boars (P < 0.05). Regression analysis revealed serum Fe concentration was positively correlated with sperm motility (r ¼ 0.251; P < 0.05), while negatively correlated with abnormal sperm rate (r ¼ 0.207; P < 0.05). However, MG and HG boars had lower serum Se concentration than LG boars (P < 0.05), and serum Se concentration contribution to sperm motility varied in a quadratic manner (Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300; P < 0.01). Semen utilization rate tended to decrease with the increase of seminal plasma Pb concentration (P ¼ 0.09). Regression analysis exhibited seminal plasma Pb negatively related to sperm motility (r ¼ 0.237; P < 0.05), while positively correlated with abnormal sperm rate (r ¼ 0.237; P < 0.05). Furthermore, seminal plasma Pb was the most influential factor among trace element in serum and seminal plasma on sperm motility basing on the generalized linear model analysis (P < 0.05). Sperm motility decreased by approximately 3.47% when seminal plasma Pb concentration increased from 0 mg/L to 11.16 mg/L. In conclusion, deficiency of serum Fe reduces semen utilization rate by impairing sperm motility and morphology, whereas excessive serum Se decreases sperm motility. More importantly, the mere existence of seminal plasma Pb has more impact on semen quality than other trace elements in serum and seminal plasma in Yorkshire boars. © 2019 Published by Elsevier Inc.
Keywords: Semen quality Seminal plasma Serum Microelements Yorkshire boar
1. Introduction * Corresponding author. Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education & Key Lab of Swine Genetics and Breeding of Ministry of Agriculture, Huazhong Agricultural University, Wuhan, 430070, PR China. ** Corresponding author. Department of Animal Nutrition and Feed Science, College of Animal Science and Technology, Huazhong Agricultural University, Wuhan, 430070, PR China. E-mail addresses: [email protected] (S. Jiang), [email protected]. edu.cn (J. Peng). https://doi.org/10.1016/j.theriogenology.2019.04.002 0093-691X/© 2019 Published by Elsevier Inc.
Essential trace elements serve as structural components of body tissues, constituents of body fluids and various enzymes in major metabolic pathways and are indispensable for physiological reproductive function [1]. However, the accumulation of nonessential potentially toxic trace elements in the body also adversely affects mammalian male reproductive system [2e4]. Therefore, maintaining reasonable addition of essential trace elements and
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avoiding toxic trace elements are important for male reproductive system. Trace elements of Cu, Fe, Zn, and Se play a key role on metabolism of carbohydrates, proteins and lipids and they have equal, beneficial or detrimental effects, depending on their balance, on mammalian male reproductive functions [5e8]. A lower level of Cu enhances sperm membrane lipid peroxidation and sperm forward motility, but excess of Cu causes marked inhibition of these biochemical parameters [9]. Seminal plasma Fe relates to sperm progressive motility, gross motility and viability in water buffalo [6]. In regard to Se, it acts as an antioxidant through various selenoproteins viz. glutathione peroxidase, thioredoxin reductase and selenoprotein P. Se deficiency and to a lesser extent Se excess both lead to decreased expression pattern of cJun and cFos genes in testicular germ cells which might be responsible for decreased germ cell number, reduced fertility, and disrupted spermatogenesis [10]. Pb and Cd, which is pervasive in environment and accumulates in the body during lifetime (especially Pb) are toxic elements for humans and other mammals [11]. The presence of Pb and Cd in seminal plasma decrease sperm motility by increasing free radical formation as well as antioxidants damage [12,13]. Although trace element test in seminal plasma is frequently used because of the importance for sperm metabolism, function and survival, that in serum is also an important indicator of testicular germ cells function and spermatogenesis in mammalian males [8,14]. However, few studies were conducted to combine trace element test in both serum and seminal plasma for semen quality analysis. In addition, it is reported the absorption and metabolism of trace elements are different among cow breeds [15]. Our previous study has demonstrated serum Cu, Fe, Mn and seminal plasma Cd levels affect semen quality of Duroc boars [16], but relationship between trace element and semen quality in other boar breeds is not clear. We hypothesized trace elements in serum and seminal plasma both can be marks of semen quality, and relationships between trace element and semen quality in Yorkshire boars are different from that in Duroc boars. Therefore, the objectives of this study were to: 1) test trace elements levels (Ca, Mg, Cu, Fe, Zn, Mn, Se, Cr, Pb, and Cd) in serum and seminal plasma; 2) analyze relationships between trace elements and semen quality using regression model; 3) determine the most influential factor among trace element in serum and seminal plasma on semen quality using generalized linear model (GLM) in Yorkshire boars. 2. Materials and methods All animal handling protocols were approved by the Animal Care and Use Committee of the College of Animal Science and Technology, Huazhong Agricultural University (Committee of Science and Technology). 2.1. Study population To evaluate the relationships between trace element level in serum and seminal plasma and semen quality, 112 healthy Yorkshire boars aged from 9 to 42 mo were selected at Yaji Mountain AI Center of YangXiang Joint Stock Company, Guangxi province, China. The Yorkshire boars were raised in stalls (0.79 by 2.40 m), which were fully slatted with concrete slats (90 mm solid width, 23 mm slots). The ambient temperature inside the house was maintained at around 19.8 Ce25.4 C and extra light was provided when the natural photoperiod could not meet the daylight regime of 12 h of lighting. The Yorkshire boars were restricted to 2.5 kg/d of commercially feed, which was the same as the diet in our previous study [16]. In addition, The Yorkshire boars were provided access to water ad libitum.
89
2.2. Semen quality assessment and experimental grouping of boars Semen sample was collected by the gloved-hand technique with a frequency of three times every two weeks during experiment period. For semen collection, sperm-rich ejaculate fractions were collected and the gelatinous fraction was strained from the ejaculate through 4 layers of cotton-mesh gauze. After sampling, four basic semen parameters were examined. Briefly, semen volume was measured by weighing each ejaculate and considering 1 g of semen to be equal to 1 mL. Sperm concentration was tested three times for each semen sample by using a sperm density meter (Fujihira Industry Co., LTD, Tokyo, Japan) and the average value was considered as the final concentration. To determine sperm motility, a computer-aided sperm analysis system (CASA system, MLe210JZ, Nanning SongJinTianLun Biological Technology Co., LTD, Nanning, China) was used. The CASA system was loaded with the MAILANG sperm automatic analytical software, and conjugate high-contrast sperm optical imaging lighting device and MAILANG computer sperm scanning counting board. The ejaculates were diluted (1:1) immediately with the isothermal Beltsville Thawing Solution (Jin Li Livestock Equipment Co., LTD, Wuhan, China). A 10-mL aliquot of semen sample was placed in a pre-warmed (37 C) microscope slide and covered with a cover glass. The grey level was set between 30 and 50, and minimum and maximum diameters were set between 3 and 20 mm in black mode when sperm motility was analyzed for five fields. To examine sperm morphology, the reagents were prepared by dissolving 0.67 g of eosin Y and 0.9 g of NaCl in 100 ml of purified water and adding 10 g of nigrosin the 100 ml of eosin Y solution firstly. Then a 10 mL aliquot of semen with an equal volume of eosinenigrosin suspension was mixed and a smear on a glass slide was made for air drying. Finally, at least 200 spermatozoa was evaluated in each replicate for each slide at a magnification of 400 under bright field optical microscope in order to achieve an acceptably [17]. Sperm with head defects, neck and mid-piece defects, tail defects, and excess residual cytoplasm were considered unnormal in this study. Total sperm number (volume concentration) and functional sperm number [total sperm number motility (1 e abnormal sperm rate)] were calculated according to Smital et al. (2004) and Wolf and Smital (2009) [18,19]. Boar semen with agglutinated sperm, sperm motility lesser than 70%, or abnormal sperm rate higher than 20% was considered as not available based on the cut-off values recommended by previous studies (normal sperm morphology 80% and sperm motility 70%) [16,20]. Therefore, the border between the nontoxic or toxic effect of elements on sperm motility and abnormal sperm rate were set at sperm motility of 70% and abnormal sperm rate of 20%, respectively. Semen utilization rate was calculated basing on semen quality of each ejaculate. According to semen utilization rate, 112 Yorkshire boars were allocated into three groups: low utilization rate group (LG, < 60% utilization rate, n ¼ 16), medium utilization rate group (MG, 60e80%, n ¼ 28), and high utilization rate group (HG, > 80%, n ¼ 68). 2.3. Sample collection and element concentrations analysis Serum and seminal plasma samples from 112 Yorkshire boars were collected in September 2016 after the semen quality of each boar was determined. Blood samples were collected by venipuncture after an overnight fast of at least 12 h, blood was allowed to clot and serum was recovered after centrifugation at 1500g for 10 min at room temperature. Sperm samples were collected at the same time as blood samples, and further centrifugation was performed to obtain seminal plasma samples. Serum and seminal plasma samples were stored at 80 C until trace element analysis.
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In addition, diet and drinking water samples of boars were also collected and stored at 4 C for trace element analysis. The method for determining the content of serum and seminal plasma elements were described in our previous study [16]. Briefly, frozen serum and seminal plasma samples were liquefied and the samples were digested in 10 mL polyethylene pipe in a fume cupboard with a 1:2 solution of highly purified HNO3 under moderate heating conditions (80 C) for about 4 h until the solution was transparent. The samples were diluted with Milli-Q water after cooling to room temperature, then these samples were mixed and filtered for determination of Ca, Mg, Cu, Fe, Zn, Mn, Se, Pb, and Cd. In addition, diet sample was pretreated by wet digestion. Drinking water sample was filtered and directly detected for element concentration. The element content in the sample was detected by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900, Agilent Technologies, Tokyo, Japan). Argon was used as the carrier gas and the isotopes 44Ca, 24Mg, 63Cu, 56Fe, 64Zn, 55Mn, 78Se, 52 Cr, 208Pb, and 111Cd were selected as analytical masses in the ICPeMS normal sensitivity mode. A Collision Reaction Interface was used for the measurements of Ca, Fe and Se to reduce common polyatomic interferences. 2.4. Statistical analysis All analyses in this study were completed in SAS (version 9.4; SAS Inst. Inc., Cary, NC). Firstly, ShapiroeWilk's test was used to verify normality of semen traits and element concentrations. Squareeroot or natural log (ln) transformed was used to correct when these parameters were not normal distribution. Indicators such as sperm motility, abnormal sperm rate, and serum Mn concentration still showed skewed distributions (nonenormal) after data transformed. Therefore, results were presented as mean ± SD for parameters with normal distribution and median for data with skewed distribution in the present study. Secondly, significant differences in semen traits and element concentrations among three groups were tested using oneeway ANOVA (normal distribution) or KruskaleWallis (skewed distribution). Thirdly, linear regression model and nonlinear regression model were carried out to explore the quantitative relationship between semen quality and element concentrations in serum and seminal plasma. Finally, the GLM, which considered semen traits as dependent variables and
element concentrations as independent variables was performed to explore the most influential factor among trace elements in serum and seminal plasma on semen quality. Briefly, element concentrations were divided into five groups in an increasing order and analyzed as classified variables. Sample sizes of the five groups were 22, 22, 22, 23, and 23, respectively. The equations of sperm motility (model 1) and abnormal sperm rate (model 2) were presented as follows. The significance level was set at 5% in all statistical analyses. Yklmn¼m1þBk þ Tl þ Sm þ Mn þ eklmn
(model 1)
Yln¼m1þTl þ Mn þ eln
(model 2)
where the Yklmn (model 1) was the value of the given sperm motility for the kth serum Cu, lth serum Fe, mth serum Se, and nth seminal plasma Pb; the Yln (model 2) was the value of the given abnormal sperm rate for the lth serum Fe and nth seminal plasma Pb; Bk is the effect of the kth serum Cu (k ¼ 1, 2, 3, 4, 5); Tl is the effect of the lth serum Fe (l ¼ 1, 2, 3, 4, 5); Sm is the effect of the mth serum Se (m ¼ 1, 2, 3, 4, 5); Mn is the effect of the nth seminal plasma Pb (n ¼ 1, 2, 3, 4, 5); and eklmn and eln were the residual effects of model 1 and model 2, respectively. 3. Results Table 1 shows that the contents of Cu, Fe, Zn, Mn, Se and Cr in diet were 17.78 mg/kg, 271.63 mg/kg, 295.71 mg/kg, 83.36 mg/kg, 0.32 mg/kg, and 1.61 mg/kg, respectively. Toxic elements of Pb and Cd were observed in diet and their concentrations were 0.14 mg/kg and 0.03 mg/kg, respectively. However, these two toxic elements were not found in drinking water. Semen quality parameters and serum and seminal plasma element concentrations of Yorkshire boars in different semen utilization groups are presented in Table 2 and Table 3. LG boars had lower functional sperm number, lower sperm motility and higher abnormal sperm rate than MG boars and HG boars (P < 0.01), and MG boars had lower sperm motility and higher abnormal sperm rate than HG boars (P < 0.01). Furthermore, as compared to HG boars, LG boars had lower serum Fe concentration (P < 0.05) and higher serum Se concentration (P < 0.05). Semen utilization rate
Table 1 Concentration of elements in diet and drinking water of boars.
Diet, mg/kg Drinking water, mg/L a
Ca
Mg
Cu
Fe
Zn
Mn
Se
Cr
Pb
Cd
7600.03 3043.94
2069.02 1806.13
17.78 Nb
271.63 N
295.71 38.34
83.36 0.31
0.32 0.12
1.61 N
0.14 N
0.03 N
recommended amount of mineral elements for boars in NRC (2012). N: the element detection content is less than 0.
b
Table 2 Semen quality parameters of Yorkshire boars in different semen utilization groups (n ¼ 112)a.
Semen volume, mL Sperm concentration, 106 mL1 Total sperm number, 109/ejaculate Functional sperm number, 109/ ejaculate Sperm motility, % Abnormal sperm rate, % a
Low utilization group (<60%, n ¼ 16)
Medium utilization group (60%e80%, n ¼ 28)
High utilization group (>80%, n ¼ 68)
Pvalueb
226.34 ± 70.85 253.40 ± 79.96 56.58 ± 23.35 35.99 ± 19.84B
263.15 ± 71.57 277.14 ± 80.55 66.79 ± 18.46 52.05 ± 18.72A
252.36 ± 70.70 274.9 ± 62.38 67.14 ± 17.08 52.87 ± 13.72A
0.25 0.52 0.13 <0.01
77.80 [66.70e82.34]C 23.46 [18.49e32.25]A
85.00 [82.96e87.34]B 13.06 [11.79e16.77]B
87.45 [86.47e88.84]A 10.07 [8.85e11.61]C
<0.01 <0.01
The results are presented as mean ± standard deviation for normal distribution or median [quartile 25- quartile 75] for skewed distribution. Different superscripts within a row indicate significant differences. P < 0.01.
b
A,B,C
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Table 3 Serum and seminal plasma element concentrations of Yorkshire boars in different semen utilization groups (n ¼ 112).a
Serum Ca, mg/L Mg, mg/L Cu, mg/L Fe, mg/L Zn, mg/L Mn, mg/L Se, mg/L Cr, mg/L Pb, mg/L Cd, mg/L Seminal plasma Ca, mg/L Mg, mg/L Cu, mg/L Fe, mg/L Zn, mg/L Mn, mg/L Se, mg/L Cr, mg/L Pb, mg/L Cd, mg/L
Low utilization group (<60%, n ¼ 16)
Medium utilization group (60%e80%, n ¼ 28)
High utilization group (>80%, n ¼ 68)
P-valueb
123.35 ± 13.35 22.41 ± 3.00 2.57 ± 0.44 1.11 ± 0.33b 0.83 ± 0.15 5.03 [0e14.90] 229.33 ± 69.57a 148.14 [110.26e199.92] 5.50 [0e11.09] 0.10 [0e0.43]
114.06 ± 16.74 22.94 ± 4.21 2.46 ± 0.59 1.15 ± 0.30ab 0.94 ± 0.29 0.52 [0e9.51] 189.80 ± 50.26b 170.44 [118.33e202.91] 9.11 [3.52e19.56] 0.15 [0e0.40]
114.92 ± 17.90 21.44 ± 3.38 2.33 ± 0.47 1.34 ± 0.39a 0.88 ± 0.29 2.03 [0e14.84] 190.29 ± 49.63b 160.64 [104.83e200.63] 7.02 [0.24e16.59] 0.20 [0e0.52]
0.17 0.15 0.19 0.02 0.41 0.84 0.04 0.64 0.25 0.35
32.94 ± 10.44 115.15 ± 48.91 49.16 [11.60e53.52] 0.54 [0.39e0.68] 23.88 ± 14.22 5.68 [1.34e9.59] 76.97 ± 27.41 47.68 [12.13e104.86] 5.44 [0.44e10.60] 0 [0e0.49]
30.90 ± 12.61 114.05 ± 59.55 44.16 [24.48e51.72] 0.44 [0.17e0.80] 22.51 ± 14.91 7.11 [2.44e10.35] 64.46 ± 30.96 53.16 [21.76e90.00] 1.66 [0e7.65] 0 [0e0.22]
29.60 ± 12.16 136.54 ± 56.84 46.87 [17.38e56.70] 0.51 [0.28e0.78] 26.64 ± 12.72 4.83 [2.21e10.18] 71.71 ± 31.25 58.71 [23.32e86.97] 0.75 [0e4.98] 0 [0e0.32]
0.65 0.23 0.92 0.76 0.44 0.82 0.49 0.95 0.09 0.61
a
The results are presented as mean ± standard deviation for normal distribution or median [quartile 25- quartile 75] for skewed distribution. Different superscripts within a row indicate significant differences. a,b P < 0.05.
b
Fig. 1. Relationships between sperm motility and the element concentrations of Yorkshire boars (n ¼ 112). (A) Sperm motility ¼ 2.441 Cu(serum) þ91.18; r ¼ 0.207, P < 0.05. (B) Sperm motility ¼ 3.349 Fe(serum) þ81.02; r ¼ 0.251, P < 0.05. (C) Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300, P < 0.01. (D) Sperm motility ¼ 0.301 Pb(seminal) þ86.21; r ¼ 0.237, P < 0.05.
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tended to decrease with the increase of seminal plasma Pb among groups. Regression analysis results of relationships between each element and sperm motility and abnormal sperm rate are summarized in Figs. 1 and 2. As shown in Fig. 1, sperm motility was negatively correlated to serum Cu and seminal plasma Pb, while positively correlated to serum Fe (P < 0.05). A quadratic relationship that increased firstly and then decreased was observed between serum Se and sperm motility (P < 0.05). As shown in Fig. 2, abnormal sperm rate decreased with the increase of serum Fe concentration, but increased with the increase of seminal plasma Pb concentration (P < 0.05). The GLM analysis was performed to determine the most influential factor among trace element in serum and seminal plasma on sperm motility. As shown in Table 4, only seminal plasma Pb showed a significant effect on sperm motility (P < 0.05), while serum Cu, serum Fe and serum Se had no significant impact on sperm motility in the GLM analysis. As shown in Table 5, sperm
Fig. 2. Variations of abnormal sperm rate with the element concentrations in Yorkshire boars (n ¼ 112). (A) Abnormal sperm rate ¼ 3.613 Fe(serum)þ17.81; r ¼ 0.207, P < 0.05. (B) Abnormal sperm rate ¼ 0.322 Pb(seminal)þ11.94; r ¼ 0.237, P < 0.05.
motility in 80e100% seminal plasma Pb group was lower than that in 20% and 20e40% seminal plasma Pb groups (P < 0.05) and it decreased by approximately 3.47% when the seminal plasma Pb increased from 0 mg/L to 11.16 mg/L (P < 0.05). Serum Fe and seminal plasma Pb had no significantly effect on abnormal sperm rate in the GLM analysis, although these two element concentrations were associated with abnormal sperm rate in the correlation analysis. 4. Discussion The relationship between trace element in serum and seminal plasma and semen quality of Yorkshire boars was investigated in this study. Results indicated serum Fe, Se and seminal plasma Pb concentrations were different among different semen utilization groups. Regression analysis revealed serum Fe concentration positively, while excessive serum Se and seminal plasma Pb concentrations negatively affects semen quality. Furthermore, GLM analysis exhibited the level of seminal plasma Pb has a greater impact on sperm motility than other influencing elements. Trace elements are important for the maintenance of normal spermatogenesis and sperm function. Fe is involved in redox process and acts as components of many enzymes and metalloprotein compounds [21]. In this study, lower serum Fe level reduced semen utilization rate by decreasing sperm motility and increasing abnormal sperm rate, which indicated serum Fe concentration may be inadequate for boars. It has been proposed sertoli cells bind and internalize diferric transferrin from blood at their basal pole, remove Fe from the basal compartment, and provide it to the meiotic spermatocytes and differentiating spermatids [22]. Serum Fe concentration limits energy production and spermatogenesis by affecting Fe metabolism in germ cells [23]. In addition, the lack of Fe affects the activity of Feedependent or Feecontaining enzymes, which increases lipid peroxidation in boar spermatozoa [24]. Therefore, inadequate serum Fe led to poor semen quality in Yorkshire boars as our previous findings in Duroc boars [16]. Se is required for vital proteins and enzymes that has important role in spermatogenesis and male fertility [25]. However, the beneficial or detrimental effect of Se on semen quality depends on its contents in body. Pipan et al. (2017) reported Se in fresh boar seminal plasma had positive correlation with sperm quality after storage [26]. However, there was no relationship between seminal plasma Se and semen quality in our study. This result may demonstrate the seminal plasma Se level in our study (70.76 mg/L), which higher than that in Pipan's study (6.21 mg/L) was sufficient for maintaining semen quality. But interestingly, we found it existed a quadratic relationship (Fig. 1C. Sperm motility ¼ 0.0004 Se2(serum) þ0.136 Seþ74.06; r ¼ 0.300, P < 0.01) between serum Se and semen quality in Yorkshire boars of the present study. Sperm motility improved as the serum Se level increased below the level of 170 mg/L (the vertex of parabola), with decreasing thereafter. Furthermore, when serum Se was higher than 367.50 mg/L (calculated basing on the equation), sperm motility decreased below 70%, which was considered as cut off value for poor semen quality. To summarize we could speculate that: 1) when serum Se level < 170 mg/L, sperm motility improved as the serum Se level increased; 2) when serum Se level ranged 170 mg/L to 367.50 mg/L, sperm motility decreased but without toxicity; 3) when serum Se level > 367.50 mg/L, sperm motility decreased below 70% with toxicity. We speculated the probable reason may be the pro-oxidant effect of excess Se, particularly in the form of selenite [27,28]. However, there was no significant correlation between serum Se and semen parameters in Duroc boars [16]. Difference in the relationship between serum Se and semen parameters in our previous [16] and this studies indicated the absorption and tolerance of Se may be different among different boar breeds. Pogge et al. (2011) demonstrated Simmental cattle had
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93
Table 4 GLM analysis the effect of multiple elements concentrations on semen quality parameters in Yorkshire boars (n ¼ 112). Parameters
Models
Results
Sperm motility
Yklm¼m1þBk þ Tl þ Sm þ Mn þ eklmn (model 1)
Abnormal sperm rate
Yln¼m1þTl þ Mn þ eln (model 2)
Source
DF
Mean square
F value
P-value
Serum Cu Serum Fe Serum Se Seminal plasma Pb Serum Fe Seminal plasma Pb
4 4 4 4 4 4
22.84 59.96 54.49 86.03 47.19 84.07
0.57 1.53 1.22 2.19 1.07 1.90
0.69 0.20 0.31 0.04 0.38 0.12
where the Yklmn (model 1) was the value of the given sperm motility for the kth serum Cu, lth serum Fe, mth serum Se, and nth seminal plasma Pb; the Yln (model 2) was the value of the given abnormal sperm rate for the lth serum Fe and nth seminal plasma Pb; Bk is the effect of the kth serum Cu (k ¼ 1, 2, 3, 4, 5); Tl is the effect of the lth serum Fe (l ¼ 1, 2, 3, 4, 5); Sm is the effect of the mth serum Se (m ¼ 1, 2, 3, 4, 5); Mn is the effect of the nth seminal plasma Pb (n ¼ 1, 2, 3, 4, 5); and eklmn and eln were the residual effects of model 1 and model 2, respectively.
Table 5 Effect of boar seminal plasma Pb on the sperm motility (model 1)a. Seminal plasma Pb
n
Concentration, mg/L
Sperm motility, %
20% 20e40% 40e60% 60e80% 80e100%
22 22 22 23 23
0 [0-0] 0 [0e0.01] 1.84 [0.75e3.48] 5.44 [4.67e7.65] 11.16 [9.60e14.74]
87.33 88.56 86.56 86.53 84.48
Statistical significance of seminal plasma Pb 20%
[86.47e89.06] [85.00e88.87] [85.53e87.94] [83.60e87.67] [81.38e87.38]
20e40%
40e60%
60e80%
80e100%
NSb
NS NS
NS NS NS
*c * NS NS
aThe results are presented as median [quartile 25- quartile 75] for skewed distribution. b NS: not significant. c * significant on the level P < 0.05.
lower plasma Se values compared to Angus cattle when calves received a same corn-silage based diet [29]. In this study, we also found serum Cu has a negative influence on sperm motility, but there was no significant difference in serum Cu among different semen utilization groups. This may be due to a slight excess of serum Cu existed in experiment boars. Olivari et al. (2009) reported high concentration serum Cu acts as a proeoxidant and leads to reactive oxygen species (ROS) production [30]. Pb and Cd are two common toxic elements for male reproductive system, and they were detected in boar diet in this study. Pb compounds initiate oxidative stress and lead to produce ROS, which causes lipid peroxidation of sperm membrane [31]. Many studies have reported total sperm number, sperm motility, sperm morphology [11,31,32], and sperm metabolism [33] are damaged by exposing to Pb. In the present study, toxic element Pb in seminal plasma showed a more significant effect on semen parameters in Yorkshire boars. This result indicated sperm exposed to toxic elements may suffer more damage than that under deficiency and excess of trace nutritional elements. Similarly, Eghbali et al. (2010) also claimed seminal plasma Pb has an adverse effect on motility and viability of spermatozoa after ejaculation in water buffalo [6]. Toxic element Cd in seminal plasma decreases sperm concentration and impairs sperm motility and morphology in human [34,35]. It possibly because Cd causes ROS production and leads to excessive protein oxidation, lipid peroxidation, and cell death [36,37]. However, only a negative correlation between seminal plasma Cd and sperm concentration was found in this study. This result of Yorkshire boars was not consistent with that reported in Duroc boars [16]. The possible reason is due to inconsistencies in absorption efficiency of toxic elements between Yorkshire boars and Duroc boars. The median of seminal plasma Cd concentration in Yorkshire boars and Duroc boars were 0 mg/L and 0.08 mg/L, respectively. We suspect Yorkshire boars are less efficient at Cd uptake or transport from blood to semen, which led to the lower level of Cd in Yorkshire boars than that in Duroc boars.
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